Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display

ABSTRACT

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein, including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of and claims priority under 35U.S.C.§ 120 to U.S. Ser. No. 09/469,194, filed Dec. 21, 1999, having thesame title and inventors, which is incorporated herein by reference.

[0002] The research carried out in the subject application was supportedin part by grants from the Department of Energy (Grant No.RDE-FG-91ER61125). The government may have rights in this invention.

INTRODUCTION

[0003] 1. Field of the Invention

[0004] The field of the invention is phycobiliprotein-assistedexpression and folding of proteins and the production of function-addedrecombinant phycobiliproteins.

[0005] 2. Background of the Invention

[0006] Many foreign proteins expressed in bacteria form insolubleaggregates called inclusion bodies. Recovery of the recombinant proteinof interest requires dissolving the inclusion bodies under denaturingconditions, removing the denaturant to allow the recombinant protein tofold, and finally purifying the recombinant protein. This process islaborious and frequently gives low yields. Inclusion bodies are formedbecause the aggregation of the newly synthesized recombinant polypeptideis faster than its folding into the native structure. In one aspect, theinvention provides bifunctional fusion constructs which are rapidlyincorporated into a macromolecular structure such that the fusionproteins are separated from one another and unable to self-associate.

[0007] In a more particular aspect, the macromolecular structures areoligomeric phycobiliproteins—a family of structurally relatedphotosynthetic accessory proteins naturally found, inter alia, incyanobacteria, the chloroplasts of the Rhodophyta (red algae) and inthose of the Cryptophyceae. When they carry covalently attached lineartetrapyrrole prosthetic groups (bilins), these proteins can exhibitbrilliant colors and intense fluorescence, making them valuable specificlabeling reagents. Unfortunately, the diversity of such reagents hasbeen restricted to naturally occurring phycobiliproteins and theirtarget specificity generated by chemical conjugation, which generatesheterogeneity (see, e.g. Siiman et al., 1999, Biocon Chem, 10,1090-1106). The use of phycobiliproteins in the invention also overcomesthese prior art limitations and provides homogenous, specific labelingreagents.

SUMMARY OF THE INVENTION

[0008] We have found that phycobiliprotein subunits are tolerant toterminal extensions of different sizes. A phycobiliprotein subunitdomain in a fusion protein is able to assemble with to cognate partnersubunit to form heterodimers, and frequently further assemble into evenhigher-order aggregates, and into the light-harvesting antennacomplexes, phycobilisomes. The non-phycobiliprotein part of the fusionprotein (the displayed domain) is exposed on the surface of theoligomeric phycobiliproteins. We define this fusion protein expressionsystem as “phycobilisome display”. Newly synthesized fusion polypeptidesare quickly separated from each other by virtue of the assembly of thephycobiliprotein domain (the carrier domain) into oligomers. Thenon-phycobiliprotein moiety (the displayed domain) of the fusion proteinis displayed on the surface of oligomer, e.g. the rods of thephycobilisome, and is able to fold into functional proteins whilesequestered from interaction with other unfolded polypeptides.Phycobilisome display can therefore be used as an alternative foldingenvironment for difficult-to-fold proteins, especially those that havebeen found difficult to fold in other organisms. The displayed proteincan be used as a fusion construct bearing a fluorescent phycobiliproteintag, or be separated from the carrier phycobiliprotein domain bycleavage of the linker peptide between the carrier domain and thedisplayed domain by a specific protease. Either embodiment may bepracticed in cells (e.g. using a resident protease) or in vitro (e.g.using purified fusion proteins. cell-free expression systems, etc.),though cleavage is preferably practiced outside the cell to control itstiming with respect to folding.

[0009] Accordingly, the invention provides methods and compositions fordisplaying a functional polypeptide domain on an oligomericphycobiliprotein. The compositions include fusion proteins comprising afunctional displayed domain and a functional phycobiliprotein domainincorporated in a functional oligomeric phycobiliprotein. In particularembodiments, the phycobiliprotein domain is a natural phycobiliproteindomain or modified variant thereof: the functional oligomericphycobiliprotein is an α, β heterodimer; the displayed domain comprisesan affinity tag, an oligomerization moiety, a specific binding moietyand/or a signaling moiety: and/or the displayed domain is refractive toexpression in E. coli. The compositions also include functionaloligomeric phycobiliproteins comprising the subject fusion proteins andcells comprising such oligomeric phycobiliproteins.

[0010] The subject methods include methods for making a functionaldisplayed domain comprising the step of combining a polypeptidecomprising a displayed domain and a phycobiliprotein domain with aphycobiliprotein subunit under conditions to form a subject fusionprotein. In particular embodiments, the methods further comprise priorto the combining step, the step of making the polypeptide by expressinga nucleic acid encoding the polypeptide: and/or after the combiningstep, the step of separating the functional displayed domain from thefunctional phycobiliprotein domain. The methods steps may occurintracellularly, e.g. in a cell which is or is a progeny of a naturalcell which naturally makes functional phycobiliprotein, or a cellengineered to produce functional phycobiliprotein.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0011] The following descriptions of particular embodiments and examplesare offered by way of illustration and not by way of limitation.

[0012] The invention provides methods and compositions for displaying afunctional polypeptide (the foreign protein or displayed domain) on anoligomeric phycobiliprotein. The compositions include fusion proteinscomprising a functional displayed domain and a functionalphycobiliprotein domain incorporated in a functional oligomericphycobiliprotein.

[0013] A functional phycobiliprotein domain is capable of assembling thefusion protein in a functional oligomeric phycobiliprotein. Preferreddomains provide at least 1%, preferably at least 10%, more preferably atleast 50%, more preferably at least 75%, more preferably at least 90%and most preferably substantially equivalent oligomer assembly abilityas that of a corresponding unfused phycobiliprotein, as measured incompetition assays, e.g. as described herein.

[0014] Any phycobiliprotein domain having the requisite functionalitymay be used and these may be derived from natural, semisynthetic orsynthetic sequences. A wide variety of natural phycobiliproteins areknown in the art, e.g. Apt and Grossman, 1995, J Mol Biol 248, 79-96.including proteins derived from many cyanobacteria, rhodophytes (redalgae) and cryptomonads. etc. (see, e.g. Glazer, 1994, J Appl Phycol 6,105-112; Glazer et al. 1995, Photosynth Res 46, 93-105), particularlyphycoerythrins, phycocyanins, and allophycocyanins. In addition, a widevariety of methods are known for modifying such natural sequences togenerate semi-synthetics. e.g. Glazer, 1994, supra, describesphycobiliproteins having non-natural, predetermined bilin compositions,and exploiting intermolecular energy transfer by functionally couplingfluorescent phycobiliprotein tags with other fluorescent tags, such ascyanine dyes, and Toole et al., 1998, Mol Micro 30, 475-486 describesrecombinations of phycobiliprotein deletion mutants. Finally, knownphycobiliprotein structure-function relationships (e.g. Anderson et al.,1998, Mol Micro 30, 467-474) are exploited to generate syntheticsequence analogs using conventional methods.

[0015] In a particular embodiment, the phycobiliprotein domain ischaracterizable as an α or β subunit, based on its sequence similarityto natural α and β phycobiliprotein subunits. The selection of α or βphycobiliprotein domains may yield different results, affecting thedisplayed domain, the carrier protein, or both, and such differencesguide the selection of the carrier phycobiliprotein subunit. Forexample, the phycocyanin a subunit and the β-L11-α subunit-fusion arepreferred when the foreign protein is displayed on the N-terminus of thecarrier protein because of the higher yield and the better spectroscopicproperties of the resulting fusion protein. For display on theC-terminus, phycocyanin β subunit and the α-L11-β subunit fusion arebetter suited because the phycocyanin a subunit is sensitive toextension on its C-terminus (usually leading to incomplete bilinaddition and in some instances partial unfolding of the protein).

[0016] In a particular embodiment, the phycobiliprotein domain confersfluorescence on the fusion protein, preferably providing fluorescencequantum yield and molar extinction coefficients at least 1%, preferablyat least 10%, more preferably at least 50%, more preferably at least75%, more preferably at least 90% and most preferably substantiallyequivalent to that of a corresponding unfused phycobiliprotein, measuredas described herein. Preferred domains provide extinction coefficientsof at least 100K, preferably at least 300K, more preferably at least 1Mand/or quantum yields of at least 0.25, preferably at least 0.5, morepreferably at least 0.6, as described herein. In other preferred aspectsof this embodiment, the fluorescence emission spectrum of the fusionprotein is substantially equivalent to that of a corresponding unfusedphycobiliprotein.

[0017] In a particular embodiment, the phycobiliprotein domain of thefusion protein comprises one or more bilins, preferably functionalbilins contributing to the visible absorption spectrum of thephycobiliprotein domain and fusion protein, preferably natural bilins.In a particular embodiment, the bilins are covalently coupled to thephycobiliprotein domain, preferably through cysteine thioether linkages,preferably at natural bilin attachment sites. The bilins may be coupledto the phycobiliprotein domain by any mechanism that provides therequisite functionalities, including enzymatic addition. Accordingly, ina particular embodiment, the phycobiliprotein domain of the fusionprotein provides a substrate for enzymatic bilin addition, which mayprovide natural or non-natural bound bilin distribution, preferably asubstrate for enzymes which naturally modify a corresponding naturalphycobiliprotein.

[0018] The displayed domain may be any polypeptide having a requisitefunctionality and being compatible with that of the phycobiliproteindomain and with assembly of the fusion protein in a functionaloligomeric phycobiliprotein. Accordingly, a wide range of displayeddomains may be used including domains comprising an affinity tag, anoligomerization moiety, a specific binding moiety and/or a signalingmoiety, and including polypeptides which may be coupled tophycobiliproteins by chemical conjugation invention (see, inter alia,U.S. Pat Nos. 4,520,110: 4,542,104; 4,857,474; 5,055,556; 5,707,804;5,728,528; and 5,869,255).

[0019] The displayed domain is preferably of length and sequencesufficient to provide a constrained structure, including at leastsecondary structure, preferably tertiary structure. In a particularembodiment, the constrained structure is of complexity sufficient torequire complex folding and is poorly expressed independent of thefusion protein in active form in conventional expression systems,particularly conventional yeast (e.g. S. cerevisiae) and bacterial (e.g.E. coli) expression systems. Preferred displayed domains are refractiveto expression when expressed independent of the fusion proteins, i.e.substantially not expressed in active form, in conventional expressionsystems, particularly E. coli. The displayed domain may have anyspectrophotometric properties compatible with the requisitefunctionalities of the fusion protein. In a particular embodiment, thedisplayed domain is substantially transparent to the wavelengths ofvisible light absorbed by phycobiliproteins, and therefore does notsubstantially affect the fusion protein and/or oligomericphycobiliprotein light-harvesting function, and/or is substantiallytransparent to the wavelength(s) of energy emitted by thephycobiliprotein domain, and therefore is not substantially affected bysuch energy.

[0020] The displayed domain may frequently be displayed on eitherterminus of the phycobiliprotein domain—important because many proteinspreferentially tolerate extension on one of their termini. Preferredexpression orientation is readily determined empirically, and in manycases (e.g. for making phycobiliprotein-labeled fluorescent reagents) isadvised by published C- and N-terminal GFP fusions. In particularembodiments, display on the N-terminals of a phycobiliprotein B subunit,rather than the α subunit, is more conducive to folding of certaindisplayed proteins. Use of the subunit-fusion phycocyanins α-L11-β andβ-L11-α as the carrier protein may also have certain advantages: thefusion proteins tend to be more stable (see, e.g. Example B, below), andthe 1:1 stoichiometry of α and β subunits is ensured.

[0021] The fusion proteins may comprise additional components asdesired, which may provide modules of functionalities, such as affinityhandles, dimer- or oligomerization domains, stabilization domains,specificity domains, signaling domains, etc., apart from any suchfunctionality/ies provided by the displayed domain. For example, forconstructs to be used as fluorescent labels, introduction of GCNoligomerization domains enhances both the spectroscopic value (morechromophores) and binding affinity (more sites for intermolecularinteraction).

[0022] In particular embodiments, the fusion protein comprises aspecific binding moiety comprising at least one of a specific bindingpair, such as a receptor—ligand pair, e.g. an immunoglobulinantigen-binding domain or antigenic domain, a lectin saccharide-bindingdomain or glycosylated or glycosylatable domain, an avidin orstreptavidin biotin-binding domain or biotinylated or biotinylatable(i.e. providing a substrate for enzymatic biotinylation) domain, etc. Ina particular embodiment, the protein comprises a biotinylated orbiotinylatable domain, which is preferably biotinylated in theexpression system (e.g. cell) selected for expression of the fusionprotein. A wide variety of synthetic, semi-synthetic and natural suchdomains are known in the art, see e.g., Schatz et al. 1993,Bio/Technology 11, 1138-1143; Tatsumi et al., 1996, Anal Biochem 243,176-180; Samols et al. 1988, J Biol Chem 263, 6461-6464, includinghomologs in phycobiliprotein producing cyanobacteria, e.g. Gornicki etal. 1993. J Bacteriol 175, 5268-5272; Phung et al., GenBank AccessionNo. U59235; Nakamura et al. 1998 Nucl Acids Res 26, 63-67. In fact,enzymes sufficient to biotinylate biotinylatable domains have beencharacterized (e.g. Beckett et al. 1999, Protein Sci 8, 921-929;Buoncristiani et al. 1988, J Biol Chem 263, 1013-1016), permitting invitro biotinylation (e.g. Li et al., 1992, J Biol Chem 267, 855-863).These biotinylated domains permit especially convenient affinitypurification tags (e.g. Cronan 1990, J Biol Chem 265, 10327-10333,Example C, below) and are useful in the many well developedbiotin/avidin applications (e.g. Wilchek and Bayer (ed) 1990. MethodsEnzymol 184, Academic Press, N.Y.).

[0023] In another example, various spacers or flexible linker peptidesproviding a variety of functionalities, such as a specific endopeptidaserecognition and/or cleavage site, an affinity-purification tag, etc.,may be used between the displayed and phycobiliprotein domains. Forexample, when displayed C-terminally to the phycobiliprotein domain, aspecific protease recognition and cleavage site can be engineeredimmediately upstream from the displayed domain so, upon cleavage withthe protease, the displayed domain can be cleanly released from thecarrier protein. This strategy also works for most proteins displayed onthe N-terminus of the carrier protein because the functions of mostdisplayed proteins are not affected by C-terminal extensions severalresidues long. In situations where such C-terminal extension is highlyundesirable, an intein domain (Perler F B, Jan 1, 2000, Nucleic AcidsRes 28, 344-345 “lnBase. the Intein Database”) can be engineeredimmediately downstream from the displayed domain. Subsequent excision ofintein cleanly releases the displayed domain from the carrier protein.

[0024] The linkers may also be used to facilitate display of domainsthat would otherwise interfere with oligomeric phycobilisome assembly.The length and amino acid sequence requirements of such functionalityare readily determined empirically for a given fusion construct.Generally, the linkers are preferably from at least 5, preferably atleast 10 residues in length, typically requiring no more than 50, andmore often no more than 30 residues. To facilitate an unintrusiveorientation (see, e.g. Example A, below) small, flexible residues suchas Ala, Gly and Ser are particularly convenient components.

[0025] The fusion proteins are incorporated in a functional oligomericphycobiliprotein. see e.g. Glazer, 1994, Adv Mol Cell Biol 10, 119-149,comprising at least a dimer, preferably an α, β, heterodimer. Theoligomers, especially when assembled into higher-order structures suchas trimers, hexamers, rods and phycobilisomes, constrain one displayedprotein from interacting with another. This is particularly useful inproducing proteins whose function is (1 ) harmful to the cell but (2)dependent on the formation of dimer or multimer. In a particularembodiment. the oligomeric fusion phycobiliproteins are structurallysubstantially identical to those of corresponding naturalphycobiliproteins and preferably provide fluorescence quantum yield andmolar extinction coefficients at least 1%, preferably at least 10%, morepreferably at least 50%., more preferably at least 75%, more preferablyat least 90% and most preferably substantially equivalent to those ofcorresponding natural phycobiliproteins. In other preferred aspects ofthis embodiment, the fluorescence emission spectrum of the oligomericfusion phycobiliproteins is substantially equivalent to those ofcorresponding natural phycobiliproteins.

[0026] Because higher order oligomers (phycobilisomes) can form acomplex with the water-splitting oxygen-evolving photosystem II, thesurrounding environment of phycobilisomes. unlike virtually all othercellular environments, may be oxidative enough for spontaneous formationof disulfide bonds. Therefore phycobilisome display may be applied tothe expression and folding of disulfide bond-containing proteins.Accordingly, the compositions also include functional oligomericphycobiliproteins comprising the subject fusion proteins and cellscomprising such oligomeric phycobiliproteins.

[0027] The fusion proteins are expressible in any convenient systemcompatible with expression of the fusion protein, preferably anempirically optimized host cell, host cell of the most closely relatednatural phycobiliprotein, or lysate or extract thereof. In a particularembodiment. the fusion protein is expressed at least 1%, preferably atleast 10%, more preferably at least 50%. more preferably at least 75%,more preferably at least 90% and most preferably substantiallyequivalent to that of a corresponding unfused phycobiliprotein domain. Awide variety of expression systems may be used. For example, Anabaenacells expressing phycobilisome displayed proteins have normal phenotypeand growth rates and can be grown large scale in defined inorganic saltmedia. Alternatively, cells or protoplasts may be engineered orreconstituted to express requisite lyase subunits (e.g. cpcE and cpcF,e.g. Fairchild et al.. 1994, Biol Chem 267, 16146-16154) and/orphycobiliprotein linker proteins (e.g. cpcC, e.g. Swanson et al., 1992,J Bact 174, 2640-2647) under conditions wherein the requisite oligomericphycobilisome proteins are formed. Reconstituted heterologous cellularsystems require either high levels of phycobiliprotein domain expression(sufficient to form dimers; see, Glazer et al., 1973, J. Biol Chem 248,5679-5685) or the expression of a compatible heterophycobiliproteindomain (to form heterodimers), i.e. simply attempting to express alabeled phycobiliprotein fusion protein with a coexpressed lyase in ahererologous cell is insufficient to form the required oligomericphycobiliproteins (Schroeder 1997, Phycobiliproteins: Biosynthesis andApplications,UC Berkeley, Dissertation). Alternatively, extracellular orcell free systems, such as lysates, extracts and reconstituted in vitrobilin addition systems may be used (e.g. Arciero et also 1988, J BiolChem 263; 18343-18349; 18350-18357; 18358-18363). Becausephycobiliproteins and phycobilisomes are made at very high levels in thecell, especially under low light intensity, phycobiliprotein displayalso helps to increase the yield of the displayed protein.

[0028] The subject methods include methods for making a functionaldisplayed domain, the method comprising the step of combining apolypeptide comprising a displayed domain and a phycobiliprotein domainwith a phycobiliprotein subunit under conditions to form a subjectfusion protein. In particular embodiments, the methods further compriseprior to the combining step, the step of making the polypeptide byexpressing a nucleic acid encoding the polypeptide: and/or after thecombining step, the step of separating the functional displayed domainfrom the functional phycobiliprotein domain. The methods steps may occurintracellularly, e.g. in a cell which is or is a progeny of a naturalcell which naturally makes functional phycobiliprotein.

[0029] Our extensive studies on phycobiliprotein subunit fusionconstructs demonstrate the versatility of phycobilisome display. Infact, our display concept may be practiced with other macromolecules,such as ribosomes, and such macromolecular complex display can also helpin posttranslational modification of proteins. For example, proteinsdisplayed on 80S ribosomes in eukaryotic cytosol can undergo differentposttranslational modifications than proteins displayed on 70S ribosomesin the mitochondria and chloroplasts. Similarly, proteins displayed ondifferent sides of the ER are also subjected to different modifications.

EXAMPLES AND DETAILED EXPERIMENTAL PROTOCOLS Example A.

[0030] Expression and characterization of recombinant phycobiliproteinfusion proteins. We describe here the expression of genes engineered toencode Anabaena sp. PCC7120 C-phycocyanin α and β polypeptides bearing a24-residue N-terminal peptide tag, incorporating a block of six Hisresidues (generally referred to as the “6xHis tag”), in wild-type andmutant cells of the filamentous cyanobacterium Anabaena sp. PCC7120. The6xHis tag allows one-step purification of the recombinant polypeptidesby immobilized metal-ion affinity chromatography [IMAC; (8)] away fromthe wild-type phycobiliproteins. We show here that phycocyanobilin (PCB)is correctly attached to the His-tagged phycocyanin α and β polypeptidesand that they fold to vield molecules whose spectroscopic propertiescorrespond to those of wild-type (α and β polypeptides.

[0031] Cultures and Strains—Escherichia coli strain DH5α (11) grown inLB medium was used in all cloning and expression experiments.Plasmid-encoded resistance to ampicillin or spectinomycin (Sp) wasselected at an antibiotic concentration of 100 μg ml⁻¹. Foroverexpression of His-tagged proteins, isopropyl-β-D-thiogalactoside(IPTG) was added to a final concentration of 0.5 mM to exponentiallygrowing cultures to derepress the trc promoter. Induced cultures wereusually grown with vigorous aeration for 5 hr at 30° C. to reduceformation of inclusion bodies. Induced cells were pelleted and stored at−20° C. until use.

[0032] Anabaena sp. strain PCC7120 and its derivative strains were grownat 30° C. on modified AA minimal medium plus nitrate (12) under medium(75 μE m⁻² s-⁻¹) to high (200 μE m⁻²s⁻¹) light intensity provided bycool-white fluorescent bulbs. Anabaena cultures overexpressingHis-tagged phycocyanin subunits were grown in half-strength AA plusnitrate medium (bubbled with 5% CO₂ in air, with 2.5 mM HEPES pH 9.0buffer to maintain pH) to late-exponential phase before addition of IPTGto 0.5 mM. The cultures were induced for 2 to 3 days before cells wereharvested and stored at −20° C. Triparental mating to introducefree-replicating plasmids into Anabaena cells from E. coli was performedessentially as described (13). Strains bearing the plasmid-borne aadAgene, encoding resistance to streptomycin (Sm) and to Sp, were selectedat 1 μg ml⁻¹ Sm plus 10 μg ml⁻¹ Sp on agar-solidified medium and at 20μg ml⁻¹ Sp alone in liquid medium. Mutant strains B646, B64328, andB64407 generated by transposon insertion (7) were selected with neomycinsulfate (Nm) at 200 μg ml⁻¹ on solid medium and 10 μg ml⁻¹ in liquidmedium.

[0033] Cloning of Anabaena sp. PCC7120 C-Phycocyanin α and βsubunits—Standard procedures were used for most molecular biologicalmanipulations (14). Genomic DNA was isolated from Anabaena sp. PCC 7120as described (15). The cpcA and cpcB genes encoding the α and β subunitsof phycocyanin, respectively, were amplified for genomic DNA by thepolymerase chain reaction (PCR; 16). The 0.5-kb PCR fragment wasdigested with the restriction enzymes NdeI and HindIII, and cloned intoNdeI- and HindIII-digested cloning vector pUC 19 [(17); GenBankaccession No. X02514], giving plasmid pBS185. The 0.5-kb PCR fragmentwas digested with enzymes NdeI and EcoRI, and cloned into NdeI- andEcoRI-digested pUC19, giving plasmid pBS251. All fragments generated byPCR were sequenced to verify fidelity using the PRISM 373 DNA sequencingsystem and the dye-terminator cycle-sequencing kit from AppliedBiosystems (Foster City, Calif.). DNA and amino-acid sequence analyseswere performed with the programs Editbase (Purdue Research Foundationand USDA/ARS) and Lasergene (DNASTAR Inc., Madison, Wis.).

[0034] In verifying the sequences of PCR products, two discrepancieswere discovered near the end of the published cpcB DNA sequence from thesame organism (5). Base number 487 (numbering of the published sequence)is an A instead of a T. This changes the Cys codon (TGC) to a Ser codon(AGC). Another difference is within the tandem CTG repeats shortlybefore the stop codon. There are four such CTG triplets, rather thanonly three shown in the published sequence. In addition to sequencingPCR-generated clones, the base changes were also confirmed by sequencingof a restriction fragment containing the entire cpcB gene. The last 36base pairs of the cpcB gene sequence determined here, encompassing bothcorrections, encode a 12-amino acid sequence identical to that ofanother filamentous cyanobacterium, Mastigocladus laminosus [(18);GenBank accession No. M75599], and the sequence of the entire CpcBpolypeptide aligns much better with those of other C-phycocyanin βsubunits (19). The mass of the Anabaena sp. PCC7120 β subunit is inexcellent agreement with that predicted from the corrected cpcBsequence. A 16.3-kb DNA sequence with the corrected cpcB sequence, thecomplete sequence of cpcF (see below), and other new information hasbeen deposited into GenBank under accession No. AF178757.

[0035] Construction of expression vectors—A family of plasmids wasspecifically constructed for inducible overexpression of His-taggedpolypeptides in both E. coli and Anabaena sp. PCC7120. Plasmid pBS150vcontains the following components: (A) From 0 to 1.3 kb, a portion frompBR322 [(20); GenBank accession No. V01119] that contains the ColE1 oriVfor plasmid replication in E. coli, the oriT (bom) site for conjugaltransfer, and the rop (repressor of primer) gene. Although the first 11codons of the Rop open reading frame were changed in pBS150v, themodified Rop protein apparently can still form the homodimerfour-alpha-helix structure (21), helping to maintain the plasmid at amedium copy number like that of pBR322 (20); (B) From 1.3 to 2.3 kb,most of the C.S4 cassette (22) containing the engineered aadA gene thatconfers resistance to Sm and Sp; (C) From 2.3 to 4 kb, a portion fromthe expression vector pPROEX-1 (Life Technologies, Inc.) that containsthe lacIq gene coding for the lac repressor, and multiple cloning sitesfor construction of sequences coding for His-tagged proteins, expressionof which is controlled by the trc promoter. The N-terminal 24 residuescontain the 6xHis affinity tag as well as a 7-amino acid recognition andcleavage site for the tobacco etch virus (TEV) protease for removal ofthe 6xHis tag if so desired (23, 24); and (D) From 4 to 4.6 kb, aportion from the expression vector pMAL-c2 (New England Biolabs, Inc.)that contains the lacZ^(α) gene for blue/white screening of insertrecombinants, and downstream, two strong, bidirectional, ρ-independenttranscriptional terminator structures from the E. coli rrnB gene (25).The nucleotide sequence of pBS150v has been deposited in GenBank underthe accession No. AF177932.

[0036] A portion of the plasmid pDU1 (26) coding for a replicationprotein and a resolvase was inserted in the unique Eco47III site ofpBS150v, giving plasmid pBS150. This 3.7-kb fragment, pDU1HC, containsspontaneous mutations that enable autonomous replication of cognateplasmids in higher copy number in Anabaena sp. (27). Plasmid pBS152v(sequence also available from GenBank under accession No. AF177933) issimilar to pBS150v but sacrifices the lacZ^(α) gene for more cloningsites like those in pPROEX-1. The pDU1HC⁺ version, pBS152. wasconstructed like pBS150. His-tagged constructs were usually made usingthe small, “v” versions of the plasmids and then converted to thelarger, pDU1HC-containing form. When induced, the trc promoter initiatesvery strong transcription in both E. coli and Anabaena. While verytightly controlled in E. coli, the trc promoter is poorly repressed inAnabaena sp. partially due to the atypical promoter and the presence ofsome codons unfavorable to Anabaena sp. in the lacI^(q) gene, leading toa relatively high level of constitutive expression of the 6xHis-taggedproteins. Preliminary Western analyses showed only about three-foldincrease in production of His-tagged proteins in Anabaena cultures uponinduction with IPTG.

[0037] Isolation of phycobilisomes—Phycobilisome (PBS) preparations werecharacterized on discontinuous sucrose density gradients as described(28), with minor modifications. Frozen or freshly harvested Anabaenacells were used with no obvious difference in results. Na/K-PO₄ buffer(0.75M; 1:1 ratio of Na-PO₄ and K-PO₄. pH 7.5) containing 1 mM each ofNaN₃ and ethylenediamine tetraacetate (EDTA), was used. Individual PBS,rod, and hexamer fractions collected from the sucrose gradients weredialyzed extensively against buffer 0 (20 mM Tris-HCl pH 8.0, 50 mMNaCl, 50 mM KCl) to eliminate sucrose, phosphate, azide, and EDTA beforeisolation of His-tagged phycobiliproteins on a Ni²⁺-NTA column (seebelow). Similarly, for isolation of intact His-tagged PBS on Ni²⁺-NTA,the PBS fraction was dialyzed against 0.75 M phosphate buffer, pH 7.5(without EDTA and azide) prior to loading on a Ni²⁺-NTA column (seebelow).

[0038] To isolate PBS containing His-tagged phycocyanin subunits, thePBS preparation in 0.75 M phosphate was loaded on 1 ml of Ni²⁺-NTA resinpre-equilibrated with 0.75 M phosphate buffer. The high concentration ofphosphate, along with possibly lesser steric availability of the 6xHistags of phycocyanin subunits incorporated within PBS, significantlydecreased the rate of binding of PBS to the Ni²⁺-NTA resin. Severalpassages of the same sample through the column were needed to getadequate binding. The resin was then washed with 20 column volumes of0.75 M phosphate buffer plus 30 mM irnidazole. PBS bound on resin wereeluted with 0.75 M phosphate buffer plus 200 mM imidazole. The eluatewas immediately dialyzed against 0.75 M phosphate buffer to removeimidazole.

[0039] Isolation of His-tagged Proteins by Immobilized Metal AffinityChromatogriaphy—Cell pellets were thawed and resuspended in 10 to 20volumes of cold (0-4° C.) buffer 0. Phenylmethylsulfonyl fluoride (PMSF)and β-mercaptoethanol were added to give final concentrations of 1 mMand 10 mM, respectively, immediately before breakage of cells by passagethrough a French press cell, three times at 18,000 p.s.i. Cellulardebris was then removed by centrifugation at 4° C. in an angled rotor at30,000×g for 20 min (preparations from E. coli) or at 130,600×g for 1 hr(preparations from Anabaena sp.). The supernatant was loaded on a columnof 1 to 3 ml of Ni²⁺-NTA resin pre-equilibrated with buffer 0. The resinwas then washed consecutively with ten column volumes each of coldbuffer A1 (20 mM Tris-HCl pH 8.0. 50 mM NaCl, 50 mM KCl, 20 mMimidazole, 5% v/v glycerol), buffer B (20 MM Tris-HCl pH 8.0, 500 mMNaCl, 500 mM KCl), and buffer A2 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50mM KCl, 30 mM imidazole). His-tagged proteins were eluted from the resinwith buffer C (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM KCl, 200 mMimidazole). The eluate was immediately dialyzed against 50 mM Tris-HClpH 8.0, 50 mM NaCl, 50 mM KCl. 1 mM DTT, to remove imidazole which athigh concentrations is slightly denaturing to proteins. RecombinantHis-tagged phycocyanin isolated in this manner consisted mostly of αβheterodimers. A crude estimate of the yield of His-tagged phycocyaninwas calculated from the amount of phycobiliprotein (expressed in unitsof A_(620 nm) ml) eluted with buffer C vs. the total amount ofphycobiliprotein applied to the Ni²⁺-NTA resin.

[0040] Size Exclusion Chromatography-High Performance LiquidChromatography—Although IMAC-purified and dialyzed samples (see above)could be used with no obvious effect on the results, proteins to beanalyzed by SEC-HPLC were generally dialyzed again in 50 mM Na-PO₄ pH7.0, 50 mM NaCl, 0.5 mM DTT. SEC-HPLC separations were performed with aWaters 600 pump and line detection achieved with a Waters 991 photodiodearray detector (Waters Associates, Milford, Mass.). A mobile phase of 50mM Na-PO₄ pH 7.0 was flowed at 0.8 ml min⁻¹ through Bio-Sil 250 (Bio-RadLaboratories, Hercules, Calif.) guard column (80×7.8 mm) and column(300×7.8 mm) maintained at 25° C. For analytical separations, 50 to 200μg of phycocyanin was loaded [based on absorbance at the peak near 621nm and using a molar extinction coefficient of 290,000 M⁻¹ cm⁻¹ permonomer (29)]. Molecular size calibration utilized bovine gammaglobulin, 158 kDa; chicken ovalbumin, 44 kDa; horse myoglobin, 17 kDa;vitamin B12, 1.35 kDa (Bio-Rad gel filtration standards). The voidvolume was determined with blue dextran, 2000 kDa (Pharmacia Biotech,Piscataway, N.J.). Preparative separations were performed with loads of0.5 to 1 mg phycocyanin.

[0041] Analytical Ultracentrifugation—Measurements were performed on aBeckman XLA centrifuge using an An-60Ti rotor maintained at 20° C. and20,000 rpm. Affinity-purified samples were run at a concentration of 0.5mg ml⁻¹ in dialysis buffer. Data were fit to a two component regression(which was superior to a single-component fit) and the molecular weightsdetermined with the HID4000 software.

[0042] SDS-Polyacrylamide Gel EIectrophoresis—Proteins were precipitatedwith 10% (w/v) trichloroacetic acid, washed once with ice-cold water,and resolubilized in SDS loading buffer [2% sodium dodecyl sulfate, 50mM Tris-HCl, pH 6.8, 100 mM DTT, 0.1% Bromphenol Blue. and 10% v/vglycerol; (13)]. SDS-PAGE (30) was performed using 10% acrylamidestacking, and 14% separating gels, with monomer/bis ratio of 37.5:1.Bilin-bearing polypeptides were visualized as fluorescent bands by UV(>312 nm) illumination after staining with 10 mM zinc acetate (31).Polypeptides were also visualized by staining with Coomassie BrilliantBlue. Protein molecular weight standards were purchased from Bio-RadLaboratories.

[0043] Absorbance and Fluorescence Spectrometry—Absorbance spectra wereacquired on a computer-controlled, dual-beam λ6 UV/V isspectrophotometer (Perkin-Elmer Corp., Norwalk. Conn.). Absorbance ofAnabaena cell suspensions was measured from 400 to 750 nm with a lightbeam passing through the frosted side of a 1-cm light path squarecuvette. Fluorescence measurements were performed, on samples with amaximal absorbance of less than 0.07 to minimize the inner-filtereffect, with a Perkin-Elmer MPF-44B fluorimeter coupled with an I/Oboard to a Macintosh computer for digitization and storage of data.Excitation and emission slits were set at 5 or 6 nm for allmeasurements. Excitation spectra were measured with emission observed ata wavelength 10 nm to the red of the sample's fluorescence emissionmaximum.

[0044] Fluorescence quantum yield measurements, for samples isolatedfrom SEC-HPLC, were made relative to cresyl violet (in ethanol,Φ_(f)=0.59; Eastman Kodak Co., Rochester, N.Y.) using the K2multifrequency phase and modulation fluorimeter (ISS, Champaign, Ill.)with excitation and emission slits set at 8 nm. All samples were dilutedto peak absorbance between 0.01 and 0.05, excited at 570 nm and emissionacquired from 575 to 800 nm. These emission spectra were theninstrument-corrected, converted to wavenumber scale, and bandpasscorrected by multiplying emission intensity at each wavelength by thesquare of the respective wavelength (32). The resulting spectra wereintegrated and quantum yields calculated according to previouslydescribed equations (33).

[0045] Molar extinction coefficients were determined according topreviously established methods (34). In brief, absorbance spectra ofsamples isolated as trimers (unless otherwise noted) from SEC-HPLC weremeasured. These samples, having peak absorbance between 0.9 and 1.2.were diluted 10× with 8 M urea, pH 1.9 containing 10 mM DTT. Theabsorbance was measured at 660 nm for these denatured samples, and molarextinction coefficients were calculated for the original native samplesfrom the known value of 35,400 M⁻¹ cm⁻¹ (at 660 nm) for eachpeptide-bound PCB in acid urea (35). Calculations assumed astoichiometry of one PCB per α subunit and two PCBs per β subunit. Molarextinction coefficient of each peptide-bound PCB at 280 nm was measuredusing trimeric phycocyanins purified from SEC-HPLC. Samples in 50 mMNa-PO₄ pH 7.0, with peak absorbance between 1.0 and 1.5 at 620 nm, weredenatured with 9 M urea to give final 8 M urea, pH 2.0. The extinctioncoefficient of a protein-bound PCB at 660 nm was identical to the valuementioned above, while that at 280 nm was calculated by subtracting,contributions from Tyr [ε_(280 nm)=1,370 M⁻¹; (36)] and Trp[ε_(280 nm)=5,500 M⁻¹ cm⁻¹; (37)] residues.

[0046] Mass Spectrometry—For electrospray mass spectrometry, purifiedproteins (0.5 to 1 mg) were dialyzed extensively against 10 mM ammoniumacetate prior to lyophilization. Proteins redissolved in 50%acetonitrile in 0.2% aqueous formic acid were analyzed with a VG Bio-Qmass spectrometer as described (38). Mass spectral analysis of differentphycobiliprotein polypeptides, as well as of some other proteinsexpressed in Anabaena sp., showed quantitative posttranslational removalof the N-terminal Met in Anabaena sp. PCC7120. For this reason. aminoacid residue numbering starts from the residue after the initial Met,and follows that used for numbering of bilin-linked residues incrystallographic studies of the highly homologous phycocyanin fromMastigocladus laminosus (39). However, our examination ofphycobiliproteins from other cyanobacteria, as well as amino acidsequence data published big others (40, 41), showed that suchposttranslational processing is not universal to cyanobacteria.

[0047] Molecular modeling of His-tagged C-phycocyanin—The crystalstructure of C-phycocyanin from the cyanobacterium Fremyella diplosiphonhas been solved at 1.75 Å, resolution (42). The structural data for thatphycocyanin, which is highly homologous to that of Anabaena sp. PCC7120,were used to build a molecular model of the Anabaena C-phycocyanin withthe 24-amino acid N-terminal extension on the α subunit. Examination ofthe C-phycocyanin hexamer structure revealed a groove lying on the βface between β subunits. In order to present a reasonable graphicalrepresentation of the HTα:β model, we chose to lead the 24-amino acidextension from inside to outside of the ring structure of phycocynintrimer through this surface groove. Although structural data supportingthis choice is lacking, our results suggest that this is a plausibleplacement. Modeling of the 24-residue extension onto the C-phycocyaninhexamer was performed using the published monomer coordinates [(42); PDBaccession code: lcpc]. The 24-residue tag was built onto the N-terminusof the α subunit as follows. First, the coordinates for a well-defined25-residue peptide (the last one at C-terminus being Met) were excisedfrom the published coordinates of another protein (antibody 48G7; PDBaccession code: IGAF). Next, all side chains except for that of the lastMet were truncated to Ala (or left as Gly). Using the least-squaressuperposition of the program LSQMAN (43), the C-terminal Met of the25-mer peptide was overlaid with the N-terminal Met of the α subunit.The tag was then coarsely laid into the above-mentioned groove using thetorsion commands of the program O (44), and the side chains mutatedagain to conform with sequence of the 24-residue tag of HTα. Again,using only the torsion commands, phi, psi, and chi angles on the24-residue tag were manipulated so as to conform to the followingconstraints: (a) all non-bonded contacts involving tag-to-tag andtag-to-phycocyanin atoms must be in excess of 2.5 Å; (b) large part ofthe tag must lay more or less in the β-face groove; and (c) the six Hisresidues must place externally to the hexamer so as to besolvent-accessible. The model was then subjected to 200 rounds of Powellenergy minimization using the program XPLOR (45, 46), during which allof the original C-phycocyanin atoms were constrained from movement. Thisresulted in the torsion angles and non-bonded contacts of the 24-residuetag approaching canonical values. Quality of the model with respect tophi/psi angles was verified using the program PROCHECK (47). TheRamachandran plot showed that the modeled tag contained no disallowedphi or psi angles. To build trimers, symmetry operators were applied tothe HTα:β monomer model using program O. The hexamer model was built byadding the tag coordinates to the second α subunit coordinates of thelcpc structure and then applying the symmetry operators.

[0048] Nomenclature—A His-tagged protein preparation purified by IMACmay contain more than one protein species. For example, both theapoprotein and holoprotein version of a His-tagged phycocyanin subunitmay be present. Consequently, we refer to such a preparation by anomenclature which specifies the organism in which it is being expressed(i.e., Anabaena sp. or E. coli), and the number of the pBS plasmid whichcontains the gene encoding the recombinant subunit. Thus, whenHis-tagged phycocyanin α subunit is expressed in Anabaena sp. PCC7120bearing the plasmid pBS168, the fraction purified by IMAC is designatedAn168. A fully characterized fraction, shown to consist of a singlespecies, for example, pure His-tagged phycocyanin α subunit, isdesignated HTα. A mutant polypeptide, for example, a His-tagged Anabaenasp. PCC7120 phycocyanin α subunit with a Thr residue in place of an Alaresidue at position 12,is designated HTα^(A12T). The absence or presenceof the bilin chromophores is indicated by the prefixes “apo” and “holo”,respectively. A His-tagged α subunit with native β subunitnon-covalently associated is designated HTα:β.

[0049] Table 1 summarizes the properties of each of the Anabaena sp.PCC7120 wild-type and derivative strains and of expression vectorscarried by these strains used for the production of His-taggedC-phycocyanin normal and mutant α and β subunits.

[0050] Expression of His-Tagged C-Phycocyanin α Subunit in Wild-TypeAnabaena sp.—The 0.5-kb NdeI-cpcA-HindIII fragment from pBS185, encodingthe wild-type phycocyanin α subunit, was cloned into pBS150, givingplasmid pBS168 encoding the α subunit with a 24-residue N-terminalextension. Cultures of Anabaena sp. PCC7120(pBS168) expressing6xHis-tagged phycocyanin α subunit were very similar to wild-typecultures grown under identical conditions with respect to the relativeamounts of phycobiliproteins and chlorophyll, and showed no apparentnegative phenotypic characteristics.

[0051] Isolation and characterization of HTα—When cell lysatesupernatant from Anabaena sp. PCC7120(pBS168) was passed through aNi²⁺-NTA affinity column, some 30 to 40% of the cell phycobiliprotein(as estimated from A_(mα) at 620 nm) was retained on the column and theneluted with imidazole, a His-tag competitor. SDS-PAGE analysis of theHis-tagged protein fraction showed two bands of 21.9 kDa and 19.5 kDa,corresponding to the calculated molecular weights of His-taggedC-phycocyanin α and native β subunits, respectively. Upon Zn²⁺-staining,both bands showed red fluorescence with near-UV excitation, indicatingthat each carried covalently linked phycocyanobilin. Identification ofthe two polypeptides was confirmed by mass spectrometry, which showedtwo components of 20,791.4±2.3 (holo-HTα minus the N-terminal Met) and19,441±1.3 (holo-β minus the N-terminal Met) in a 1:1 ratio. These massvalues confirm the presence of one PCB on HTα and two PCBs on the βsubunit. No other signiticanii peaks, especially that corresponding toan apo-HTα, were present in the mass spectrum. These data indicate thatthe protein purified by IMAC, An168, is phycocyanin holo-HTα:holo-β.

[0052] The assembly forms of phycocyanin holo-HTα:holo-β and theirapparent molecular weights were determined by analytical SEC-HPLC. Asshown in Table 2, three components were present under our particularchromatographic conditions, a monomer (calculated mass 40.5 kDa) at 49.2kDa, a trimer (calculated mass 121.5 kDa) at 145.3 kDa, and a hexamer(calculated mass 243 kDa). Over 90% of the protein was trimeric,(HTα:β)₃, the major assembly state observed under these conditions fornative PC isolated from wild-type Anabaena sp. PCC7120. The SEC-HPLCdata were corroborated by analytical ultracentrifugation, where most ofthe HTα:β was found to have a mass of 125.9 kDa, and the balance a massof 34.8 kDa (Table 2). All three fractions from SEC-HPLC were shown bySDS-PAGE to contain holo-HTα and holo-β in a ratio of 1:1. Thus, theAn168 His-tagged protein preparation purified by IMAC consists of HTα:β,and displays aggregation behavior typical of native PC under similar invitro conditions.

[0053] (HTα:β)₃ isolated by SEC-HPLC had a λ_(mα) of 621 nm at proteinconcentrations >0.5 mg ml⁻¹, and gave spectra with blue-shifted maxima(to as low as 615 nm) upon dilution (Table 3). This is consistent withthe blue shift in λ_(mα) of wild-type phycocyanin when higher orderassemblies dissociate to monomers as the protein concentration islowered (48). Interestingly. at high protein concentrations, HTα:βdisplays a λ_(max) slightly red-shifted relative to that of native PCmeasured at similar concentrations. The ε_(M) of (HTα:β)₃ was determinedto be 9.21×10⁵ M⁻¹ cm⁻¹ at 621 nm, similar to values observed for nativephycocyanin from Anabaena sp. and other cyanobacteria [Table 3; (49)].The excitation spectrum for 650 nm emission coincided nearly perfectlywith the absorbance spectrum, indicating that the HTα:β preparationbehaved as a single species with respect to spectroscopic properties.The λ was at 642 nm (with 560 nm excitation) with a Φ_(F) of 0.22,similar to a value of 0.27 measured for native Anabaena PC (Table 3). Insummary, the spectroscopic characteristics of HTα:β are virtuallyidentical to those of native Anabaena sp. PCC7120 C-phycocyanin.

[0054] Incorporation of HTα into Phycobilisomes—Since holo-HTα:holo-βrepresents between 30 and 40% of the total phycobiliprotein in Anabaenasp. PCC7120(pBS168), it appeared likely that holo-HTα is effectivelyincorporated into phycobilisomes. This possibility was examined bitanalyzing phycobilisomes and their partial dissociation products for thepresence of holo-HTα. Compared to the wild-type Anabaena sp. PCC7120phycobilisome preparation, in the Anabaena sp. PCC7120(pBS168)preparation a lower percentage of the total phycobiliproteins was foundin the phycobilisome fraction, while more was found in the rodsubstructure and the hexamer fractions. This indicates that thephycobilisomes of the strain expressing holo-HTα are less stable underthe conditions used for phycobilisome preparation. SDS-PAGE analysisshowed that holo-HTα was present in all three fractions.

[0055] Anabaena sp. PCC7120(pBS168) phycobilisomes loaded on a Ni²⁺-NTAcolumn (in the 0.75M Na/K-PO₄ pH 7.5 buffer needed to maintainphycobilisome integrity, see “Materials and Methods”), bound to thecolumn, although the binding capacity of the column under theseconditions appeared low. The His-tagged phycobilisomes were eluted athigh imidazole concentration. After removal of imidazole by dialysis,the SDS-PAGE polypeptide profile and spectroscopic properties of thisphycobilisome fraction were very similar to that of the startingphycobilisome preparation. These results show that the 6xHis tags of theHTα subunits incorporated into phycobilisomes are relatively exposed,available for interaction with the Ni²⁺-NTA matrix.

[0056] Expression of His-Tagged C-Phycocyanin β Subunit in Wild-TypeAnabaena sp.—The 0.5-kb NdeI-cpcB-EcoRI fragment from pBS251, encodingthe wild-type phycocyanin β subunit. was cloned into pBS150, givingplasmid pBS262 encoding the β subunit with the 24-residue N-terminalextension. Cultures of Anabaena sp. PCC7120(pBS262) expressing HTβ werephenotypically very similar to those of Anabaena sp. PCC7120(pBS168)expressing HTα, except for a slightly yellowish appearance. Theirwhole-cell absorbance spectra showed a small decrease in the amount ofphycobiliprotein per cell relative to chlorophyll.

[0057] The yield of the His-tagged phycobiliprotein fraction fromAnabaena sp. PCC7120(pBS262) was 16-18% of the total phycobiliprotein inthe cell lysate, substantially lower than the 30-40% in thecorresponding fraction from Anabaena sp. PCC7120(pBS168) expressing HTα(see above). Analysis of the His-tagged phycobiliprotein preparation,An262, by SDS-PAGE showed the presence of holo-HTβ and holo-α in equalamounts. Mass spectral analysis showed that the two subunits carried thenormal amounts of PCB, i.e., one PCB per α and two per HTβ subunit, andno apo-polypeptides were present.

[0058] In analytical SEC-HPLC, the α:HTβ holoprotein preparationisolated by IMAC was primarily trimeric (calculated mass 121.5 kDa) at136.9 kDa. The shoulder/tail of that peak, constituting less than 5% ofthe total His-tagged protein loaded, could be attributed to monomers(calculated mass 40.5 kDa) at 62.4 kDa (Table 2). Both fractions wereshown by SDS-PAGE to contain holo-α and holo-HTβ in a molar ratio of1:1. In contrast to native PC and the HTα:β holoprotein, the hexamercomponent was not observed for the α:HTβ holoprotein. Analyticalultracentrifugation data were consistent with two components observed inSEC-HPLC: a majority of the α:HTβ holoprotein had a mass of 121.9 kDa,and a small fraction of 43.4 kDa, corresponding closely to thetheoretical masses of trimers and monomers, respectively.

[0059] The λ_(mα) of the trimeric α:HTβ holoprotein component laybetween 615 and 619 nm depending on protein concentration (Table 3). Asobserved with HTα:β, λ_(mα) shifted blue upon dilution. However, at highprotein concentration the λ_(mα) of the α:HTβ holoprotein, like that ofnative PC, did not exceed 619 nm, in contrast to the λ_(mα) of 621 nmobserved for HTα:β under similar conditions. The ε_(M) of α:HTβ trimerswas found to be 8.88×10⁵ M⁻¹ cm-⁻¹ at 619 nm (Table 2), a slightly lowervalue than that observed for HTα:β. The excitation spectrum for 650 nmemission corresponded nearly perfectly to the absorbance spectrum,indicating that the α:HTβ preparation behaved as a single species withrespect to spectroscopic properties. The λwas at 642 nm (with 560 nmexcitation), with a Φ_(F) of 0.23 (Table 2). Thus the spectroscopiccharacteristics of α:HTβ are virtually identical to those of nativeAnabaena sp. PCC7120 C-phycocyanin and its HTα:β counterpart.

[0060] A sucrose density gradient preparation of phycobilisomes fromAnabaena sp. PCC7120(pBS262) expressing HTβ showed distribution ofphycobiliprotein aggregates different from that of the wild-type cells.A much smaller proportion of the phycobiliprotein (25%) sedimented inthe phycobilisome fraction, with 40% and 35% in the rod and hexamerfractions, respectively. SDS-PAGE analysis showed that HTβ was presentin all three fractions. However, less HTβ (relative to the otherphycobiliproteins) was found in phycobilisomes compared to the amountfound in the products of phycobilisome dissociation, rods and hexamers,suggesting a reduced stability of phycobilisomes that have incorporatedmore HTβ subunits. As described above for Anabaena sp. PCC7120(pBS168)expressing HTα, His-tagged phycobilisomes could be isolated from theAnabaena sp. PCC7120(pBS262) phycobilisome fraction by IMAC, although ina relatively poor yield. The low yield might be due to the smallernumber of HTβ subunits present per phycobilisome, a reflection of thesmaller amount of holo-HTβ relative to holo-β produced per cell.

[0061] Highly purified (HTα:β)₃ and (α:HTβ)₃ phycocyanins were used tomeasure the extinction coefficient of peptide-bound PCBs in 8 M urea pH2.0. Two different preparations were measured for each protein, and fourmeasurements gave very similar values. The molar extinction coefficientfor one peptide-bound PCB at 660 nm is very close to the value of 35,400M¹ cm⁻¹ (35). Upon subtraction of contributions from Trp and Tyrresidues, the extinction coefficient for one peptide-bound PCB at 280was determined to be (14.85±0.1)×10³ M⁻¹ cm⁻¹ (Table 3).

[0062] Expression of HTα and HTβ in a cpcBAC background—The separateoverexpresslion of HTα and HTβ in phycocyanin-minus backgrounds wasexamined in mutant strain B646 (7). This mutant has a transposoninserted between the promoter and the CpcB open reading frame, which isexpected to eliminate transcription of cpcBAC, genes encoding the , andα subunits of phycocyanin and a rod linker polypeptide, respectively(5). Strains B646(pBS168) and B646(pBS262) (see Table 1), generated byintroduction of pBS168 and pBS262 into strain B646 by conjugation, werephenotypically indistinguishable from the parent mutant strain. Bothphycocyanin-deficient strains grow slowly and produce very low levels ofthe His-tagged polypeptides.

[0063] SDS-PAGE analysis of the His-tagged phycobiliprotein fractionsfrom strain B646(pBS262) showed no α or β subunits of wild-typephycocyanin. However, multiple attempts at purifying the HTα from strainB646(pBS168) yielded fractions containing some native β subunit,presumably resulting from a basal leaky transcription of the cpcBACoperon.

[0064] On SEC-HPLC fractionation, the IMAC-purified proteins fromB646(pBS262). An262-BAC, eluted as a single peak of subunit homodimers,i.e., (HTβ)₂. Purified native β subunits of C-phycocyanin dimerize atconcentrations of 1-10 μM (34, 50). Since mass spectrometry showed thatthe HTβ subunits produced in this cpcBAC background carried a fullcomplement of PCB, it is evident that bilin addition to phycocyanin βsubunit is independent of the presence of the phycocyanin apo- or holo-αsubunit. The mass spectral analysis also indicates the presence in HTβof the posttranslational methylation at the γ-amino group of the Asn⁷²residue normally found in phycocyanin β subunits (51, 52).

[0065] Under similar SEC-HPLC conditions IMAC-purified proteinpreparation from B646(pBS168), An168-BAC, fractionated into threespecies: trimer, monomer, and subunit. SDS-PAGE analysis of thefractions indicated that the trimer and monomer fractions contained HTαand holo-β in equimolar amounts. The subunit fraction contained onlyHTα, the majority of which appeared to be apo-HTα based on the relativeintensities in the absorption peaks at 280 nm and 618 nm, and fromcomparison of Zn²⁺- and Coomassie-staining intensities of the An 168-BACtrimer and monomer fractions (HTα:β) run in parallel on the same gel.

[0066] Analyses of the His-tagged protein preparations from strainsB646(pBS168) and B646(pBS262) indicate that overexpression of HTβ in thecpcBAC background yields a soluble protein where the HTβ subunit isstabilized by homodimer formation. The results also indicate that HTαappears to be unable to form subunit homodimers in the cell, and mayrequire the presence of β subunit that is extremely limited in thecpcBAC background for stabilization. The greatly reducedchromophorylation of HTα protein isolated from this cpcBAC strain mayalso reflect a possible requirement of subunit dimerization (α:β, α:α,or β:β) prior to covalent attachment of PCB, or misfolding of asignificant fraction of apo-HTα in the absence of apo- or holo-β.

[0067] The HTα and HTβ subunits isolated from mutant strainsB646(pBS168) and B646(pBS262) have absorbance spectra and molarextinction coefficients (Table 3) similar to those reported forrenatured α and β subunits of native C-phycocyanin, obtained bychromatographic separation under denaturing conditions (34, 50).Algebraic addition of the HTα and HTβ spectra yielded a spectrum withshape similar to that of monomeric PC but with a blue-shifted maximum at611 nm, consistent with previous observations (34). Incubation of theHTα and HTβ subunits together overnight at 4° C., followed byfractionation by SEC-HPLC, yielded a trimer fraction with absorbance andfluorescence excitation and emission spectra similar to thecorresponding spectra of HTα:β, α:HTβ, and native PC. This indicatesthat holo-HTα (but not apo-HTα, see below) interacts preferentially withholo-HTβ to form trimers, and His-taggecd holophycocyanin subunitsisolated in the absence of their cognate subunit partners can bereconstituted to yield trimers with properties similar to those ofnative phycocyanin.

[0068] Expression of His-tagged phycocyanin subunits in anapophycocyanin α subunit PCB lyase-deficient background—A dimeric lyase,CpcEF encoded by genes cpcE and cpcF, specifically catalyzes theaddition of PCB to Cys⁸⁴ of the phycocyanin α subunit (1, 3). We haveobtained two mutants through transposon mutagenesis, B64328 with aninsertion in the cpcE gene, and B64407 in the cpcF gene. Both mutantshad greatly reduced cellular content of normal phycocyanin (7).Expression of His-tagged phycocyanin subunits in the mutant strainsprovided a simple way of assessing how mutations in each of the twoCpcEF lyase subunits affect PCB addition to the phycocyanin to subunit,and how cognate PC proteins behave in the cell.

[0069] Expression of HTα in either a cpcE or a cpcF background—Toexpress the HTα polypeptide in either a cpcE or cpcF background, plasmidpBS168 was introduced into both mutants, giving strains B64328(pBS168)and B64407(pBS168) (see Table 1). The two strains showed theyellowish-green phenotype characteristic of their respective parentmutants. The His-tagged phycobiliprotein fraction from either mutantstrain amounted to <5% of the total absorbance of the cell lysate at 607nm. The His-tagged protein purified from the cpcF strain B64407(pBS168),An168-F, was shown by SDS-PAGE and mass spectral analyses to beapo-HTα:holo-β, with no detectable holo-HTα. In contrast, the An168-Eprotein contained apo-HTα:holo-β along with a small amount ofholo-HTα:holo-β. On SEC-HPLC, apo-HTα:holo-β is seen to form monomerspreferentially, in contrast to the preferred trimer formation byholo-HTα:holo-β.

[0070] Sucrose density gradient sedimentation patterns of phycobilisomepreparation from strains B64328(pBS168) and B64407(pBS168) were verysimilar to those given by the parental strains B64328 and B64407 (7). Inboth cases, the phycobilisome fraction consisted largely of theallophycocyanin-containing phycobilisome cores carrying much reducedamounts of rod components (relative to phycobilisomes from wild-typecells). SDS-PAGE analysis showed the presence of apo-HTα in thephycobilisome and rod/hexamer fractions and an amount ofphycoerythrocyanin, a distal component of Anabaena phycobilisome rodsubstructures, greater than that seen in wild-type phycobilisomes (7,53). The relative amount of apo-HTα was higher in the phycobilisomefraction than in the rod/hexamer fraction, suggesting that apo-HTαassembled into phycobilisomes might be turned over more slowly in thecell.

[0071] The finding that apo-HTα:holo-β assembles into phycobilisomes isconsistent with earlier observations of such incorporation of thephycocyanin apo-α subunit in cpcE and cpcF mutants of the unicellularcyanobacterium Synechococcus sp. PCC7002 (3, 4). We showed previouslythat the Anabaena sp. PCC7120mutant strains B64328 (cpcE) and B64407(cpcF) incorporated the phycocyanin apo-α:holo-β into phycobilisomes,and that this “defective” phycocyanin still allowed the assembly intophycobilisomes of the distal phycoerythrocyanin component (7). Theexperiments reported here extend that observation to phycocyaninapo-HTα:holo-β proteins.

[0072] Expression of HTβ in either a cpcE or a cpcF background—Toexpress the HTβ polypeptide in either a cpcE or cpcF background, plasmidpBS262 was introduced into both mutants, giving strains B64328(pBS262)and B64407(pBS262) (see Table 1). Yield of His-taggedholophycobiliproteins from either mutant strain was low, accounting for<7% of the absorbance of the cell lysate at 607 nm. In contrast toresults obtained with the mutant strains expressing HTα (see above),when HTβ was expressed, SDS-PAGE analysis showed that the His-taggedprotein preparations contained apo-α and holo-HTβ in a molar ratio ofapprα imately 1:2. SEC-HPLC fractionation showed presence ofapo-α:holo-HTβ trimers and monomers, and of (holo-HTβ)₂ homodimers. Theapo-α:holo-HTβ trimers had spectroscopic properties virtually identicalto those of apo-HTα:holo-β trimers described above. Analyses ofphycobilisomes and of rod/hexamer fractions showed that some of theholo-HTβ (likely in the form of apo-α:holo-HTβ) was incorporated intoboth fractions.

[0073] Expression of His-tagged mutant phycocyanin subunits—Thefavorable expression and incorporation of His-tagged wild-typephycocyanin subunits in Anabaena sp. provides a convenient means toperform in vivo and in vitro studies of mutant phycobiliproteinsubunits. Here we present two examples.

[0074] Expression of HTα^(A12T)—One of the cpcA clones generated by PCRusing the Taq DNA polymerase was found to have a single G ->A basechange leading to an Ala¹²-> Thr mutation in the translated protein. TheAla¹² residue is conserved in all sequenced phycocyanin α subunits (19).The crystal structure of phycocyanin predicts that replacement of Ala¹²by a residue with a larger side chain would interfere with anheterodimer formation by steric hindrance of the interaction of α-Asp¹³with β-Tyr⁹⁷ (54). We expressed the HTα^(A12T) mutant subunit inwild-type Anabaena sp. PCC7120 to examine this prediction.

[0075] The mutant cpcA gene was cloned into pBS150, giving plasmidpBS167. Anabaena sp. PCC7120(pBS167) expressing HTα^(A12T) wasphenotypically very similar to strain Anabaena sp. PCC7120(pBS168) (seeTable 1). However, the His-tagged protein fraction was obtained in vervpoor yield from Anabaena sp. PCC7120(pBS167):<0.15% of thephycobiliproteins in the cell lysate as estimated from A_(620 nm). Whilespectroscopically similar to the An 168 His-tagged protein preparation,mass spectral analysis of the An167 His-tagged protein preparationshowed two components in similar amounts, with masses corresponding tothose of holo-α and holo-HTα^(A12T). These results lead to twoconclusions. First, the mutant HTα^(A12T) subunit appeals to have agreatly reduced affinity for the holo-β subunit. Most of the HTα^(A12T)is likely to be present as free subunits in the cell and to be degradedrapidly. HTα^(A12T) also seems to have a low affinity for holo-α, and isconsequently stabilized in vivo by the formation ofholo-α:holo-HTα^(A12T) dimers. Second, the in vivo covalent attachmentof PCB to the phycocyanin α subunit evidently does not require that itform a complex with the β subunit.

[0076] Sucrose density gradient preparation of phycobilisomes fromstrain Anabaena sp. PCC7120(pBS167) gave a banding pattern similar tothat from the wild type. The HTα^(A12T) subunit was not detected inphycobilisome and rods fractions, suggesting that HTα^(A12T) was notassembled into the phycobilisomes (although the very low amount ofHTα^(A12T) may have evaded detection).

[0077] Expression of HTβ^(S46G,N76D)—One of the cpcB fragments,PCR-amplified using the Taq DNA polymerase, contained two mutations:both an A -> G change, in bases number 139 and 229, respectively, of thepublished sequence (5). The changes resulted in the replacement ofresidue Ser⁴⁶ with Gly, and of residue Asn⁷⁶ with Asp in the CpcBprotein. Anabaena sp. PCC7120(pBS162) expressing the HTβ^(S46G,N76D)mutant PC subunit was phenotypically identical to the one expressingHis-tagged wild-type β subunit. The HTβ^(S46G,N76D) mutant proteinbehaved the same way as the His-tagged wild-type counterpart in allaspects tested (yield, assembly states in vitro, assembly intophycobilisome in vivo, etc.) except that the purified protein, An162,had an absorption maximum at 610 nm, about 8-nm blue-shifted compared tothat of the An262 protein. Fluorescence emission maximum of the An162protein, however, was still at 642 nm.

[0078] Ser⁴⁶ of the phycocyanin β subunit, situated in the coil regionconnecting helices A and B (39, 54), is not in contact with anychromophore. Its non-conserved substitutions include Gly in somephycobiliprotein subunits. It therefore may be assumed that theSer⁴⁶->Gly change would not have obvious effects on the protein. TheAsn⁷⁶ residue, on the other hand, is highly conserved amongmulti-chromophore β subunits of phycobiliproteins (19), and itssidechain is in contact with ring D of the α-84 PCB chromophore of PCtrimers, possibly involved in maintaining its stretched confirmation intrimer aggregates (55). The neighboring Arg⁷⁷ residue. also highlyconserved, forms a hydrogen bond with the propionic acid substituent ofring B of the β-84 PCB (54, 55). In the mutant protein, replacement ofAsn⁷⁶ by Asp⁷⁶ sidechain may interfere with that hydrogen bonding,thereby affecting absorbance of β-84 PCB. A reduction of absorbance ofβ-84 PCB would shift the λ_(mα) of the cognate phycocyanin to the blue(50).

[0079] Molecular modeling of His-tagged C-phycocyanin—Based oncrystallographic data obtained from the cyanobacterium Fremyelladiplosiphon C-phycocyanin (42) that is highly homologous to that ofAnabaena sp. PCC7120, a molecular model was built for the Anabaena sp.PCC7120 C-phycocyanin incorporating the 24-residue N-terminal extension.In this particular model, the N-terminal extension can be led frominside to outside of the ring structure of the phycocyanin trimer,through a groove between β subunits on the β side of the trimer. Thisallows unhindered α face-to-α face stacking of two trimers to form ahexamer. The 24-residue N-terminal tag, occupying only the groove spacein the β faces, also does not appear to interfere with stacking ofhexamers on β faces to form rods of the phycobilisome. The firstN-terminal 13 of the 24 residues of the tag, that include the six Hisresidues, are completely exposed outside of the rod surface, consistentwith the observed affinity purification of phycobilisomes by IMAC (asdescribed above).

[0080] In summary, methodology developed in this study allows (a) highlevel expression of holo-HTα and holo-HTβ in Anabaena sp. PCC7120 and avariety of its mutant derivatives; (b) facile affinity purification ofthe His-tagged polypeptides and of complexes into which they areincorporated, including intact phycobilisomes; (c) study of in vivoassembly and bilin addition: and (d) analysis of site-specific mutantsof C-phycocyanin. It is evident that the approaches described here areimmediately generalizable to other Anabaena phycobiliproteins, such asallophycocyanin and phycoerythrocyanin (3, 7, 29).

[0081] With appropriate choice of organism, these approaches can beapplied to the expression and study of other phycobiliproteins includingphycoerythrins. Finally, the studies described here provide for thedesign and expression of diverse fusion proteins in Anabaena sp. withimportant outcomes for the successful production of modular fluorescentphycobiliprotein tags and for the exploration of heterologous proteinfolding in cyanobacteria (9, 10).

Example B.

[0082] Recombinant phycobiliprotein fusion proteins with oligomerizationand biospecfic recognition domains. Here we describe the design andexpression of more complex recombinant phycobiliprotein constructs whichincorporate oligomerization and biospecific recognition domains and insome of which the α and β subunits are covalently bridged. Materials andmethods essentially as described in Example A are not restated.

[0083] Assays of protein binding to streptavidin—Western hybridizationwas carried out essentially as described (14). Proteins separated onSDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membrane(Immobilon; Millipore Corp., Bedford, Mass.) using a mini Trans-Blotcell (BioRad Laboratories). Electrophoretic blotting was carried out inice-cooled transfer buffer for 1 hr at 100 V. The PVDF membrane was thenblocked with 3% bovine serum albumin in PBS buffer (115 mM NaCl, 4 mMKH₂PO₄, 16 mM Na₂HPO₄, pH 7.4) plus 0.5% Tween-20 (Sigma Chemical Co.).Binding of membrane-bound proteins to streptavicdin was assayed byincubating the membrane at 25° C. for 1 hr in PBS buffer plus 0.1%Tween-20 and 1:5000-diluted streptavidin-alkaline phosphatase conjugate(Prozyme Inc., San Leandro, Calif.). Alkaline phosphatase-mediated colordevelopment on the membrane using chromogenic substrates5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium(NBT) was carried out as described (14) except that 200 mM Tris-HCl pH8.8 was used as the developing buffer.

[0084] Alternatively, streptavidin-coated agarose beads were used totest binding of Strep2-tagged phycocyanin. As has been described (56),beads coated with recombinant core streptavidin (Biometra Inc., Tampa,Fla.) gave better results than those coated with natural streptavidin(Sigma Chemical Co. or Prozyme Inc.). Streptavidin-coated beads wereincubated at 4° C. for about 20 min with excess amount of purifiedStrep2-tagged phycocyanin, washed twice with buffer W (100 mM Tris-HClpH 8.0, 1 mM EDTA), and visualized by fluorescence. Phycocyanin-labeledbeads were observed either in batch with ultraviolet (>312 nm)excitation of fluorescence viewed through a 550 nm long-pass filter, orunder an epifluorescence microscope (the BH-2 system from OlympusAmerica Inc., Lake Success, N.Y.) with excitation light through a450-480 nm band-pass filter and emission observed through a 515 nmlong-pass filter.

[0085] Construction of modular cloning vectors for protein expression—Aseries of cloning anal expression vectors was designed and constructedfor expression of fusion proteins with different functional domains andtags on the N-terminus. To facilitate shuffling of the functionaldomains to produce desired combinations, DNA cassettes coding for thesedomains were designed as exchangeable modules. Care was taken in thedesign to ensure that only optimal codons (in respect to E. coli andAnabaena sp. PCC7120) were used, and that no restriction sites (for 6-bpcutters) were present in the core sequences, except for designed-insignature sites.

[0086] The expression vector pBS150v [Example A; GenBank Accessionnumber AF177932] was used as a template in the engineering of functionaldomains. Generally, a pair of designed oligonucleotide primers was usedto run inverse PCR, producing a new plasmid in which the sequence frombp 3892 to 3938 (encoding the 6xHis affinity tag and a spacer) ofpBS150v is replaced by a desired functional module.

[0087] Replacement with the 56-bp NcoI-BspMII Strep2 module generatedplasmid pBS283v (4,644 bp; Table 4). The 10-residue Strep2 tag is ableto bind specifically to streptavidin (57).

[0088] Replacement with the 146-bp NcoI-AgeI combination module (6xHisplus GCN4-p11) gave plasmid pBS311 v (4,734 bp; Table 4), and with the164-bp combination module (Strep2 plus GCN4-pLI) gave pBS303v (4,752 bp;Table 4). Sequences for the GCN4-pII and GCN4-pL1 coiled coil domainswere modified from part of the original Saccharomyces cerevisiae GCN4sequence encoding the leucine zipper domain (58, 59), to conform withthe pII sequence (positions a and d are both Ile) or the pLI sequence(positions a and d are Leu and Ile, respectively) (60). A BglII site wasengineered into the GCN4-pII coding sequence as a signature site, as wasan AsuII site in GCN4-pLI.

[0089] With plasmids pBS283, pBS303, and pBS311 in hand, four functionaldomains, 6xHis, Strep2, GCN4-pII, and GCN4-pLI, are available forfurther manipulation. Since the different functional domains are modularin design, different cloning and expression plasmids with variouscombinations of the modules were easily made by simple recombinationcloning (Table 4). All resulting plasmids have the modular region endwith the second half of either the BVspMII or the AgeI site, encoding aGly, immediately upstream from the recognition and cleavage sequence forthe tobacco etch virus (TEV) endoprotease (Example A, herein, 23, 61).The 7-residue TEV site was retained in all plasmids to act as a spacerbetween the modular functional domains and the multiple cloning sitesinto which genes of interest are inserted, and to provide a means ofremoving the modular functional domains from expressed fusion proteinsif so desired. The LacZ^(α) domain downstream from the TEV site wasfound to be functional in all cognate plasmids, allowing blue/whitescreening of inserts in the multicloning region. The multicloning regionof these plasmids can be greatly enhanced in lieu of the LacZ^(α)function by recombination with plasmid pBS152v [(Example A, herein);GenBank Accession number AF177933]. For plasmids encoding phycocyaninsubunits, the NdeI-cpcA-HindIII and NdeI-cpcB-EcoRI fragments were takenfrom plasmids pBS185 and pBS251, respectively (Example A, herein).

[0090] Construction of genes encoding phycocyanin subunit-fusionmonomers—X-ray crystallographic data obtained from the highly homologousC-phycocyanin from another cyanobacterium Fremyella diplosiphon (42)were used to design subunit fusions of the Anabaena phycocyanin, seeExample A. Straight-line distance between α carbons of the last residueof α subunit and the first residue of β subunit was measured to be 29.5Å. The distance between the last residue of β subunit and the firstresidue of a subunit was 32.6 Å. Although the average sum of threechemical bonds' length of a peptide unit is 4.3 Å, the actualstraight-line distances between two α carbons were found to range from3.2 to 3.7 Å among several peptide units measured in the crystalstructure. An 11-residue linker was therefore chosen to link theC-terminus of one subunit to the N-terminus of another. The 11-residuelinker is sufficiently long to bridge the distance (29.5 to 32.6 Å)between the N- and C-termini of the two subunits, and provides someleeway for bending should this be required for higher-order assembly ofthe resultant fusion phycocyanins.

[0091] One obvious difference between phycocyanins of Fremyelladiplosiphon and of Anabaena sp. PCC7120 is the lack of the initial Metresidue in the Anabaena subunits as a result of posttranslationalmodification (Example A, herein). To reduce possible steric interferencein the engineered Anabaena protein, an Ala was inserted in place of themissing Met at the N-terminus of the subunit following the fusionlinker. Since both α and β subunits have an α-helical C-terminal regionmade up mostly of small hydrophobic residues, Ala was chosen as thefirst residue in the fusion linker to maintain the local hydrophobicenvironment. The rest of the linker peptide (L11) was designed for highstructural flexibility, and consists mostly of Gly residues and two Ser.Incorporation of the signature site XmnI in the L11 coding sequence notonly facilitates characterization of PCR products, but also allows easyconstruction of fusions of other proteins to the C-terminus ofphycocyanin α or β subunits (Example C, herein).

[0092] Designed oligonucleotide primers incorporating the L11 linkersequence were used for inverse PCR with template DNA consisting of bothpBS185 and pBS251 plasmids (Example A. herein), giving plasmids pBS310(NdeI-cpcA-L11-cpcB-EcoRI in pUC19) and pBS307(NdeI-cpcB-L11-cpcA-HindIII). Once sequenced to confirm fidelity, the1047-bp NdeI-EcoRl fragment of pBS310 was cloned into NdeI- andEcoRI-digested pBS150 (Example A, herein), giving plasmid pBS320 whichencodes 6xHis-tagged CpcA-L11-CpcB fusion phycocyanin. In like mannerpBS315 was made to encode the 6xHis-tagged CpcB-L11-CpcA fusion protein(Table 4). A PCR-generated spontaneous mutant product, in which 6 basesin the signature XmnI site of the L11 linker sequence had been deleted(giving the L9 linker), was also used in this study (pBS3419; see Table4).

[0093] All other molecular biological manipulations followed standardprocedures (14). To ensure sequence fidelity, all DNA fragments that hadgone through the PCR process (16) were sequenced using the PRISM 373 DNAsequencing system and the dye-terminator cycle-sequencing kit fromApplied Biosystems (Foster City, Calif.). Analyses of DNA and amino-acidsequence data were performed with the programs Editbase (Purdue ResearchFoundation and USDA/ARS) and Lasergene (DNASTAR Inc., Madison, Wis.).

[0094] Nomenclature—A recombinant polypeptide encoded by an expressionplasmid is referred to by the plasrnid number, and the His-taggedprotein fraction isolated by affinity chromatography is designated bythe organism in which the recombinant polypeptide is expressed. Forexample, a phycocyanin a subunit with both 6xHis and Strep2 tags encodedby plasmid pBS327, expressed in E. coli and purified by IMAC isdesignated Ec327. The purified. characterized protein is then designatedas HT-Strep2-α. The same construct expressed in Anabaena sp. PCC7120 isdesignated An327. However, the purified protein is HT-Strep2-α:β,because in Anabaena sp. the HT-Strep2-α forms a stable complex with theholophycocyanin β subunit. Phycobiliproteins expressed in E. coli areapoproteins (lacking attached phycobilins), whereas those expressed inAnabaena sp. are holoproteins, unless otherwise noted.

[0095] Design of subunit-fusion phycocyanins—A His-tag at the N-terminusof either an α or a β subunit does not interfere with the folding ofthese polypeptides into their native structures (Example A, herein). Twopossible α/β fusions can be generated with an 11-residue (L-11) linker:HTα-L11-β and HTβ-L11-α. In either construct, one subunit, the α or theb, would have a 24-amino acid extension (including the 6xHis tag) at theN-terminus and a large fusion, linked through L11, at its C-terminus.Both constructs were made to determine the folding and spectroscopicproperties of such fusions.

[0096] Expression of His-tagged phycocyanin β-α fusion protein in E.coli—HTb-L11-α is expressed in E. coli as the apoprotein (lacking thethree phycocyanobilins). At 37° C., in cultures induced with IPTG for 5hrs, the fusion protein (Ec315) represented over 5%, of total cellularproteins (>7.5 mg per liter of culture at 10⁹ cells ml⁻¹), and at 30°C., in cultures induced with IPTG for 12 hr, some 20% of total cellularproteins. HTβ-L11-α purified by IMAC could be concentrated up to 0.2 mMprotein (nearly 8 mg ml⁻¹) in 50 mM Tris-HCl pH 8.0, 50 mM NaCl. 50 mMKCl, 1 mM DTT, and 5% glycerol. The yield and solubility were markedlyhigher than that obtained upon expression of the individual His-taggedphycocyanin subunits, HTα (Ec 168) and HTβ (Ec262), in E.coli (ExampleA, herein). With IPTG induction at 30° C., in the latter cases, yieldswere about 0.1 mg per liter of culture. Upon IPTG induction at 37° C.,˜60% of the individually expressed 6xHis-tagged subunits remained insolution while the balance formed inclusion bodies. Thus coexpression ofthe apo-α and apo-β subunits, inherent in the expression of HTβ-L11-α,evidently promotes folding and concomitant retention of solubility ofthe recombinant apoprotein.

[0097] Expression of His-tagged phycocyanin β-α fusion protein inAnabaena sp.—Cultures of Anabaena sp. PCC7120(pBS315) expressingHTβ-L11-α appeared more blue than that of the wild type. Whole-cellabsorbance spectra showed a much higher ratio of phycocyanin:chlorophylla. Despite appearing very healthy, Anabaena sp. PCC7120(pBS315) culturesgrew about 30% more slowly than the wild type.

[0098] When cell lysate supernatant from Anabaena sp. PCC7120(pBS315)was passed through a Ni²⁺-NTA affinity column, usually >33% of the totalphycobiliproteins (as estimated from A_(620 nm)) was retained on thecolumn and then eluted with imidazole (resultant protein denoted An315).Such high yields are comparable to that of the strain expressing HTα(>30% of A_(620 nm)), and better than that of the strain expressing HTβ(˜16%) (Example A, herein).

[0099] SDS-PAGE analysis showed that the An315 fraction consisted almostentirely of HTβ-L11-α, along with a small amount (<10%) of nativephycocyanin α and β subunits in a ˜1:1 ratio. This finding suggests thatthe fusion construct interacts relatively normally with unmodifiedphycocyanin in the cell. Such interaction to form higher assemblies issupported by analysis of Anabaena sp. PCC7120(pBS315) phycobilisomes,which were indeed found to contain HTβ-L11-α. Much more of the fusionprotein was found in the phycobilisome substructures (rods andhexamers/trimers) than in intact phycobilisomes, suggesting thatphycobilisomes with a higher content of HTβ-L11-α may be less stableduring the preparation procedure. The high amount of HTβ-L11-α relativeto native phycobiliproteins in the hexamer/trimer fraction also suggeststhat the construct is turned over more slowly in the cell. In thiscontext, it is noteworthy that the whole-cell phycobiliproteinfluorescence emission of Anabaena sp. PCC7120(pBS315) was twice as highas that of strains expressing His-tagged α or β subunit, and the growthmedia of this strain often turned blue, the color of phycocyanin.

[0100] On SEC-HPLC the An315 preparation fractionated into hexamers andtrimers only, with no monomers observed (Table 5). This result parallelsthose obtained previously with An168 (HTα:β) and An262 (αHTβ), where theα and β subunits are not covalently linked (Example A. herein). Thetrimer component, (HTβ-L11-α)₃, had a λ_(mα) at 622 nm at >10⁻⁶ Mprotein. (HTβ-L11-α)₃ dissociated to monomers at very low proteinconcentrations, with absorption maxima shifting blue to as low as 615 nm(Table 6), a behavior characteristic of native phycocyanin and of(HTα:β)₃ and (αHTβ)₃ holoproteins. At high dilution, HTβ-L11-α has anA_(max):A_(360 nm) of 6.2, similar to the values observed for monomericwild-type and His-tagged α:β phycocyanins (Table 6; Example A, herein).The εfor (HTβ-L11-α)₃ at >10⁻⁶ M protein concentration was determined tobe 900,000 M⁻¹ cm⁻¹. The fluorescence emission spectrum for 560 nmexcitation of HTβ-L11-α, with λat 643 nm and a Φ_(f) of 0.21, werevirtually identical to those of wild-type phycocyanin. The fluorescenceexcitation spectrum for 655 nm emission corresponded well with theabsorbance spectrum (Table 6). The above data show that the HTβ-L11-αfusion phycocyanin has properties virtually identical to those of thewild-type and His-tagged α:β holo-proteins, and indicate that HTβ-L11-αexpressed in Anabaena sp. has a full complement of bilins, i.e., one PCBper α and two per β subunit.

[0101] Mass spectral analysis of the SEC-HPLC trimer fraction,(HTβ-L11α)₃, gave a value of 40,993.2±6.8 which corresponds well to thetheoretical value (41,118.8 Dal) of the holoprotein HTβ-L11-α with theinitial Met residue (131.1 Dal) removed. The mass spectral analysis notonly confirms the full posttranslational chromophorylation on both the αand β subunit moieties. but also indicates presence of theposttranslational methylation on the γ-amino group of the Asn⁷² residueof the β subunit domain (51, 52).

[0102] Expression of His-tagged phycocyanin α-β fusion protein inAnabaena sp.—Like HTβ-L11-α apoprotein (Ec315), HTα-L11-β (Ec320) wasexpressed in E. coli in very high yield in soluble, form. The phenotypeand growth rate of cultures of Anabaena sp. PCC7120(pBS320) expressingHTα-L11-β were similar to those of strains expressing only His-tagged αor β subunits. The whole-cell absorbance spectrum of Anabaena sp.PCC7120(pBS320) was similar to that of the wild type but with a slightlyhigher and blue-shifted contribution from phycocyanin, and about 50%higher whole-cell phycobiliprotein fluorescence was observed.

[0103] Like HTβ-L11-α (An315), HTα-L11-β(An320) was also expressed atvery high yield in Anabaena sp., often accounting for >32% of totalcellular phycobiliproteins. SDS-PAGE showed that affinity-purified An320proteins were almost entirely HTα-L11-β, with a small amount ofcopurified native phycocyanin α and β subunits. The ratio of copurifiedα to β subunits was ≧2, suggesting a reduced affinity of HTα-L11-β fornative phycocyanin β subunits.

[0104] On SEC-HPLC, the An320 fraction separates into three components:monomers, trimers. and hexamers. It is noteworthy that nativeC-phycocyanin under the same chromatographic conditions was mainlytrimeric with very little monomer, whereas the HTα-L11-β preparation wasa ˜1:1 mixture of trimers and monomers (Table 5). The theoreticalmolecular weights from these components are 13, 19, and 9% higher,respectively, than the values observed (Table 5). These discrepanciesindicate that the radius of gyration of HTα-L11-β is smaller than thatof the wild-type phycocyanin αβ monomer.

[0105] At >10⁻⁶ M protein concentration, the SEC-HPLC trimer and monomerfractions of HTα-L11-β had λ_(mα) at 608 and 605 nm, respectively. Thislikely accounts for the blue-shifted phycobiliprotein contribution tothe whole-cell absorbance spectrum of Anabaena sp. PCC7120(pBS320). Likewild-type phycocyanin, the trimer fraction showed a blue shift in itsabsorbance spectrum down to 605 nm with decreasing proteinconcentration, indicative of dissociation of the trimers to monomers. Athigh dilution, HTα-L11-β has an A_(max):A_(360 nm) of 5.2, substantiallylower than the value of 6.4 observed for monomeric wild-type and HTα:βphycocyanins (Table 6; Example A).

[0106] The unusual characteristics exhibited by the HTα-L11-β proteinpurified from Anabaena sp. were similar to those of apo-HTα:β (ExampleA, herein). The trimer and monomer SEC fractions of HTα-L11-β weretherefore compared to the HTβ-L11-α holo-protein (both fusion proteinshave identical amino acid composition). When denatured in acid urea, thetrimeric fraction of HTα-L11-β had PCB absorbance about midway betweenthree PCBs per protein (holoprotein) and two PCBs per protein, while themonomeric fraction gave absorbance very close to that of two PCBs perprotein. An interpretation of that result is that the α domain ofHTα-L11-β fusion protein is not fully chromophorylated. Only about 50%of the trimeric HTα-L11-β proteins had PCB in the α domain, while mostmonomeric HTα-L11-β proteins had no PCB addition in the a domain.Because of the nearly equal distribution between trimers and monomers,fewer than 30% of the a subunit domains of affinity-purified HTα-L11-βproteins carry bilin. It is noteworthy that apo-HTα:holo-β andapo-α:holo-HTβ proteins also run mostly as monomers on SEC-HPLC, withapparent molecular weights substantially smaller than theoreticalcalculations (Example A, herein). The fact that most of the HTα-L11-βconstructs have an apo-α domain could also explain why more native αthan native β phycocyanin subunits were copurified with HTα-L11-β, sincethe α-Cys⁸⁴ bilin is involved in the α:β interaction and in stablemonomer packing (55, 62)

[0107] Electrospray mass spectral analysis of the SEC-HPLC monomerfraction of HTα-L11-β gave only one major peak with a value of40,327.8±5.7, corresponding well with the value of 40,331.7 daltons,obtained by subtracting from 41,118.8 Dal (molecular weight ofholo-HTα-L11-β) the values 131.1 (initial Met residue), 57.0 Dal (theC-terminal or the second residue from the N-terminus, Gly), 585.0 (onePCB), and 14.0 (one methyl group). This not only confirms the incompletebilin addition (likely on the α domain) of HTα-L11-β, but also suggestsimpaired posttranslational methylation on the γ-amino group of Asn⁷² ofthe β domain (51, 52). The latter is unexpected because HTβ (Example A,herein) and HTβ-L11-α (see above) purified from Anabaena sp. are bothfully methylated on β-Asn⁷².

[0108] The SEC-HPLC trimer fraction of HTα-L11-β (at >10⁻⁶M) had a εof677,000 M⁻¹ cm⁻¹. The fluorescence emission spectrum for 560 nmexcitation, with λat 643 nm and a Φ_(f) of 0.22. was virtually identicalto that of wild-type phycocyanin. The fluorescence excitation spectrumfor 655 nm emission corresponded reasonably well with the absorbancespectrum, with a slight shift in the maximum to 613 nm, likely due tothe small amount of native phycocyanin in the preparation (Table 6). TheSEC-HPLC monomer fraction of HTα-L11-β had spectroscopic propertiessimilar to those of apo-HTα:holo-β monomers (Example A).

[0109] Effect of shortening the linker—A PCR-generated mutant, HTα-L9-β,with the linker Ala-(Gly)₄-Ser-(Gly)₃, was isolated in the course ofproducing the HTα-L11-β construct, where L11 isAla-(Gly)₄-Ser-Gly-Ser-(Gly)₃. In other respects the two constructs wereidentical. Like the HTα-L11-β apoprotein (Ec320), the HTα-L9-βapoprotein (Ec319) was expressed in E. coli In high yield. HTα-L9-β(An319) isolated from Anabaena sp. exhibited the same, anomalousspectroscopic properties described above for the HTα-L11-β construct.Analysis of the HTα-L9-β protein by SEC-HPLC showed trimers and monomersin a ratio of 1:4, significantly lower than the ratio of 1:1 observedfor HTα-L11-β. Since the extent of chromophorylation of trimer andmonomer fractions of HTα-L9-β was similar to those of respectivefractions of HTα-L11-β, the reduced trimer formation by HTα-L9-β may bea result of conformation change induced by the shorter linker betweenthe subunits.

[0110] Expression of phycocyanin fusion proteins incorporatingoligomerization domains—The dissociation of phycocyanins to the monomerat low protein concentration is highly undesirable when these proteinsare used as fluorescent tags, particularly because of the resultantdecrease in the number of PCB chromophores per tag. The finding thatrecombinant phycocyanin subunits with a 24-residue extension at theN-terminus fold and assemble in the same manner as native phycocyaninsubunits (Example A, herein) suggested the introduction of the GCN4oligomerization domains (60) at the subunit N-termini to produce stablephycocyanin oligomers. Expression ofphycocyanin α subunit fused at theN-terminus to the trimerization domain GCN4 pII—The 33-residue peptideGCN4-pII forms homotrimeric parallel coiled coils with K_(D)<10⁻⁹ M(60). Plasmid pBS314 (encoding HT-pII-α) was constructed to express aGCN4-pII-CpcA fusion (Table 4).

[0111] When HT-pII-α was expressed in E.coli, the recombinant proteinwas found almost entirely in inclusion bodies. Similar results wereobserved whether the induction was at 30 or 37° C. This behavior is insharp contrast to that seen with HTα [Ec168; (Example A, herein)], wheremost of the recombinant protein remains soluble. A likely explanation isthat trimerization of the GCN4-pII in the fusion protein is faster thanthe folding of the phycocyanin subunit domain and interferes withfolding of the latter by promoting random aggregation of the partiallyfolded subunit domains.

[0112] Cultures of Anabaena sp. PCC7120(pBS314) expressing HT-pII-αshowed no negative phenotype, but had a bluer appearance than the wildtype. Whole-cell absorbance spectra of Anabaena sp. PCC7120(pBS314)showed a higher ratio of phycocyanin:chlorophyll α. Whole-cellfluorescence was ˜70% higher than that of the wild type or cellsexpressing HTa. Sucrose density gradient fractionation of Anabaena sp.PCC7120(pBS314) phycobilisome preparations showed that HT-pII-αrepresented a much higher proportion of the total phycocyanin in thetrimer/hexamer fraction than in the intact phycobilisome fraction.

[0113] Over 40% of the phycobiliprotein in Anabaena sp. PCC7120(pBS314)cell lysate supernatant bound to the Ni²⁺-NTA column and eluted with 200mM imidazole. This was the highest relative level of recombinantphycobiliprotein of any of the His-tagged constructs we have expressedin the course of these studies. An314 bound much more tightly to theNi²⁺-NTA column than His-tagged phycocyanin constructs not containingoligomerization domains. The tightness of binding was evidenced by theslow desorption of the recombinant protein from the Ni²⁺-NTA resin andthe larger volume of imidazole buffer required for elution. Suchbehavior is to be anticipated from a molecule in which threepolypeptides, each with a 6xHis tag, are trimerized through the GCN4-pIIcoiled coil. SDS-PAGE analysis showed that the An314 phycobiliproteinfraction was a stoichiometric HT-pII-α:β holoprotein complex.Chromatograplhy on SEC-HPLC showed that the HT-pII-α:β preparationwas >75% trimer with some higher molecular weight component(s),presumably hexamer. Monomers were not detected.

[0114] The spectroscopic properties of (HT-pII-α:β)₃ are compared withthose of the native C-phycocyanin trimer, (α:β)₃ in Table 6. Thetrimerized construct has significantly higher A_(max):A_(360 nm) ratiothan the native (α:β)₃ and a higher Φ_(F). Moreover, the absorbancespectrum of (HT-pII-α:β)₃ was unchanged at low protein concentrationswhere native phycocyanin, HTα:β, and α:HTβ were monomeric withsignificantly blue-shifted absorbance maxima and decreasedA_(max):A_(360 nm) ratios (Table 6; Example A, herein). Fluorescencepolarization measurements provided an independent assessment of thestability of (HT-pII-α:β)₃ at low protein concentrations. In trimericphycocyanin, the fluorescence polarization is very low because of rapidenergy transfer among the nine phycocyanobilin chromophores. Incontrast, in the phycocyanin monomer, the three PCBs are well separatedand depolarization through energy transfer is minimized. Our data showthat at high protein concentrations, where various phycocyaninpreparations are trimeric, their fluorescence polarization is similarand low, with values of 0.035 to 0.070. The fluorescence polarization ofHTα:β and α:HTβ, like that of native phycocyanin, rose sharply at verylow protein concentrations, indicative of trimer dissociation. whereasthat of (HT-pII-α:β)₃ was independent of protein concentration.

[0115] Expression of phycocyanin α subunit fused at the N-terminus tothe tetramerization domain GCN4-pLI—The 33-residue peptide GCN4-pLIforms very stable homotetrameric parallel coiled coils (60). Severalplasmids were constructed to express GCN4-pLI-α fusion proteins (Table4), including plasmids pBS321 (encoding HT-pLI-α) and pBS323 (encodingHT-Strep2-pLI-α). The 10-residue Strep2 tag was incorporated into theHT-Strep2-pLI-α to test whether it would confer streptavidin-bindingspecificity on the construct.

[0116] When expressed in E. coli, GCN4-pLI-α fusion proteins, Ec321(HT-pLI-α) and Ec323 (HT-Strep2-pLI-60 ), were mostly found in inclusionbodies. In contrast, when expressed in Anabaena sp. the fusion proteinsAn321 and An323 remained soluble. The yields of these His-taggedphycobiliproteins from this strain were high, reaching >20% of totalphycobiliprotein. As described above for the GCN4-pII-α fusion proteins,the GCN4-pLI-α fusion protein (An321) bound very tightly to the Ni²⁺-NTAresin. SDS-PAGE analysis showed that An321 protein fraction purified byIMAC had the composition of HT-pLI-60 :β. Analysis by SEC-HPLC showedtwo components, a small amount of tetramer, (HT-pLI-60 :β) ₄, and thebalance as a much larger component. The elution position of the latterlay outside the range of calibration of the size standards for thecolumn, but extrapolation of the calibration curve allowed an estimateof the size of the larger component. The calculated molecular weight wasconsistent with that of a trimer of tetramers (Table 5).

[0117] Presumably, such a trimer would be formed by the interaction ofthree (HT-pLI-α:β)₄ assemblies through one of their phycocyanin α:βdomains (while the other three α:β domains forming a trimer) in a manneranalogous to that leading to the formation of (HT-pII-α:β)₃. In such a“trimer of tetramers”, all α:β monomers are within phycocyanin trimers.Tetramers. each held together by a GCN4-pLI domain, would be theexpected products of dissociation of such a “trimer of tetramers” atvery low protein concentration. In accord with this expectation, theabsorbance spectrum of HT-pLI-60 :β at low protein concentration showeda λ_(mα) of 621 nm and an A_(max):A_(360 nm) ratio of 7.5 (Table 6),values very similar to those obtained for (HT-pII-α:β)3 (see above).

[0118] HT-Strep2-pLI-α:β(An323) exhibited physical and spectroscopicproperties identical to those of HT-pLI-60 :β (An321) (Table 6), showingthat the presence of the Strep2 sequence does not interfere withtetramerization by the GCN4-pLI domain.

[0119] Expression of subunit-fusion phycocyanins fused at the N-terminusto the trimerization domain GCN4-pII—As described above, His-tagged β-αsubunit-fusion phycocyanin (An315) has spectroscopic propertiesidentical to those of HTα:βand α:HTβ. Like those proteins, however,HT-β-L11-α also dissociates to monomers at high dilution. To increaseutility of the HT-β-L11-α fusion phycocyanin as a fluorescent label, wefused the GCN4-pII domain to this construct to enhance trimer stability.

[0120] Plasmid pBS358 encodes HT-pII-β-L11-α (Table 4). When expressedin E. coli, HT-pII-β-L11-α apoprotein was found mostly in inclusionbodies, but when expressed in Anabaena sp. the HT-pII-β-L11-α proteinremained soluble. Cultures of Anabaena sp. PCC7120(pBS358) hadphenotypes very similar to those of the Anabaena sp. PCC7120(pBS315)(see above), including the slower growth, “bluer” color, and increasedwhole-cell fluorescence. Analysis of phycobilisome preparations fromthis strain showed that HT-pII-β-L11-α was incorporated into thephycobilisomes. The 6xHis-tagged phycobiliprotein fraction (An358)purified by IMAC represented >22% of total cellular phycobilinroteins.As noted above for An314 (HT-pII-α:β) the An358 fraction also bound verytightly to the Ni2+-NTA resin.

[0121] SDS-PAGE analysis of the An358 fraction showed that itconsisted >90% of HT-pII-β-L11-α along with a small amount of copurifiedwild-type phycocyanin α and β subunits (in a ratio of 1:1). Analysis bySEC-HPLC at protein concentrations <500 μg/ml, showed thatHT-pII-β-L11-α preparation was largely trimeric with a small amount of alarger component (Table 5). Comparison of the spectroscopic propertiesof (HT-pII-β-L11 -)3, purified by SEC-HPLC, with those of nativeC-phycocyanin trimer (α,β)3, showed no significant differences (Table6). The absorbance spectrum of (HT-plI-b-L11-a)3 did not change withdilution to very low protein concentration (Table 6), indicating thatthe GCN4-pII domain prevented dissociation of the trimer.

[0122] The α-β fusion constructs purified from Anabaena sp., HTα-L9-βand HTα-L11-β, preferentially form monomers even at micromolar proteinconcentrations (see above). The similar construct incorporating theGCN4-pIl domain, HT-pII-α-L11-β, is encoded by plasmid pBS362 (Table 4).The HT-pII-α-L11-β protein purified from Anabaena sp., An362, was foundto be largely trimeric (84%) with the remainder hexameric (Table 5). Nomonomer was observed in the SEC-HPLC analysis. It is evident that thetrimeric state of HT-pII-α-L11-β depends on trimerization of theGCN4-pII domain. Although remaining trimeric at low proteinconcentrations, the HT-pII-α-L11-β protein had spectroscopic propertiessimilar to those of HTα-L11-β protein (Table 6), including theincomplete chromophorylation of the α domain.

[0123] Expression of recombinant phycocyanins with a tag conferringbiospecificity—The constructs described above successfully address therequirement that the phycocyanin constructs maintain their oligomericstate at very low proteins concentrations. Next, we examine theintroduction of an additional domain into such constructs, one whichconfers biospecific recognition. As a test case, we introduced a tagwhich binds specifically to streptavidin. Recombinant phycocyaninscontaining such a tag are immediately useful as reagents in the manyapplications of the widely used streptavidin-biotin methodologies (63).

[0124] Expression of Strep2-tagged phycocyanin constructs—The 10-residueStrep2 peptide forms complex with streptavidin with a K_(D) of 7.2×10⁻⁵M (57). It is believed that the Step2 tag retains its affinity forstreptavidin when fused either to an N- or C-terminus of a protein (57).Plasmids pBS327 and pBS323 were constructed to encode HT-Strep2-α andHT-Strep2-pLI-α. respectively (Table 4). E. coli and Anabaena strainsexpressing either fusion protein were phenotypically identical to thoseexpressing the corresponding non-Strep2-tagged proteins.Affinity-purified HT-Strep2-α:β (An327) and HT-Strep2-pLI-α:β (An323)proteins also had spectroscopic and aggregation properties very similarto corresponding non-Strep2-tagged proteins HTα:β (An168) and HT-pLI-α:β(An321) (Tables 5 and 6).

[0125] When blotted on a membrane, HT-Strep2-α (Ec327 and An327 andHT-Strep2-pLI-α (Ec323 and An323) were readily detected by probing withstreptavidin-alkaline phosphatase conjugate, indicating that the Strep2peptide sandwiched between the 6xHis tag and the GCN4-pLI domain stillretains its affinity for streptavidin. When streptavidin-coated agarosebeads were used as target, HT-Strep2-α:β holoprotein was largely removedfrom the beads on washing, a result anticipated from the low affinity ofa single Strep2 tag for streptavidin [K_(D)=7.2×10⁻⁵ M: (57)]. TheHT-Strep2-pLI-60 :β holoprotein, with its multiple Strep2 tags, boundmuch more strongly to the streptavidin-coated beads. This experimentdemonstrates that oligomeric phycobiliprotein fusions with multipleStrep2 tags show the appropriate target specificity instreptavidin-based fluorescence assays.

Example C.

[0126] Recombinant phycobiliprotein fusion proteins with carbαyl-terminal tagging.

[0127] We present here the in vivo production of biotinylatedphycocyanin constructs that are readily usable in the manywell-developed biotin/avidin applications (63). Materials and methodsessentially as described in Example A or B are not restated.

[0128] Construction of expression plasmids—Plasmids coding for relevantprotein fusion constructs are listed in Table 7. The expression vectorpBS150v [(Example A); GenBank Accession number AF177932] was used tomake plasmid pBS339v (4,677 bp) for expression to His-tagged proteinscapable of being biotinylated in E. coli. The 7 codons between the 6xHistag and the TEV site in pBS150v were replaced with the 21 codonsspecifying the BTN tag. The 13-residue sequence of the BTN tag wasderived from the consensus sequence that can be biotinylated in E. coli(64). An AsuII restriction site was designed into the BTN sequence as asignature site to facilitate PCR product characterization and subsequentrecognition of the BTN tag sequence. The NdeI-cpcA-HindIII fragment frompBS185 (Example A) was inserted between NdeI and HindIII sites ofpBS339v, giving plasmid pBS329v (5,138 bp) encoding 6xHis- andBTN-tagged phycocyanin a subunit (Table 7). The BTN tag is positioned onthe carbα yl-terminal side of the 6xHis tag, so that any recombinantmolecules that have lost the biospecificity tag through proteolysis arenot retained on the IMAC column. The NdeI-glbN-HindIII fragment fromplasmid pGlbN (66) was similarly cloned to give plasmid pBS355, encoding6xHis- and BTN-tagged cyanoglobin (Table 7). The same NdeI-HindIIIfragment cloned into pBS t50v gave plasmid pBS121v (5,005 bp) encoding6xHis-tagged cyanoglobin (Table 7).

[0129] The cloning vector pBS370v (4,589 bp) for expression ofC-terminal Strep tagged proteins (Table 7) was derived from the cloningvector pBS152v [(Example A); GenBank accession No. AF177933] usinginverse PCR. The 45-bp HindIII-BglI fragment at the end of the multiplecloning sites was replaced with the 68-bp HindIII-Strep-BglI sequence.An EheI site was engineered into the Strep tag coding sequence (56) as asignature site. Phycocyanin genes. in the form of NdeI-XmnI fragmentsfrom cognate plasmids (Example B) were inserted between NdeI and XmnIsites of pBS370v to generate phycocyanin-Strep tag fusions. Theexpression cloning vector pBS350v, an enhanced version of pBS152v, wascreated by replacing bp 3,801 to 4,070 of pBS152v with the 333-bpsequence described above.

[0130] DNA fragment encoding the 114-AA C-terminal portion of theAnabaena sp. PCC7120 BCCP protein (BCCP114) was amplified from Anabaenagenomic DNA. The 0.36-kb PCR fragment was digested with EcoT22I andcloned into the EcoT22I site of pBS350v, giving plasmid pBS344v (4,977bp). Again, phycocyanin genes, in the form of NdeI-XmnI fragments fromcognate plasmids (Example B), were inserted between NdeI and XmnI sitesof pBS344v to generate phycocyanin-BCCP114 fusion constructs with theBTN flexible linker. The 20-residue flexible linker between the twomoieties was designed to (a) bear a thrombin recognition and cleavagesite to allow separation of the fusion partners if so desired, and (b)be sufficiently long to allow packing of BCCP 114 on the outside of therod substructures after the fusion protein is assembled inphycobilisomes (Example A, Example B). The GCN4-pII trimerization domainwas introduced via modular cloning (Example B) between the 6xHis tag andthe TEV protease site N′ of the phycocyanin genes.

[0131] Expression of BTN-taggedfusion proteins—The 13-residue BTN taghas the consensus sequence of peptides found to be biotinylated in E.coli (64). Covalent attachment of a biotin to the Lys residue isapparently catalyzed by the biotin ligase encoded by the birA gene (65,67). The BTN tag was fused to the N-terminus of the phycocyanin αsubunit in an attempt to produice in vivo biotinylated phycocyanin. Whenthe HT-BTN-α phycocyanin subunit, encoded by plasmid pBS329, wasexpressed in E. coli, the purified Ec329 protein was shown biotinylatedin Western analysis. The same protein produced in Anabaena sp. PCC7120(An329), however, was very poorly biotinylated, if at all, even when theculture medium had been supplemented with as much as 500 μM biotin. Twoother assays for the presence of biotin on An329, competition with HABA{2-[(4′-hydrα yphenyl)-azo]benzoic acid} for streptavidin in solution(68) and mobility shift in SEC-HPLC, indicated lack of biotinylation.Parallel results were obtained in a comparison of Ec317 (HT-BTN-pII-α)and An317 (HT-BTN-pII-α:β) proteins.

[0132] Sucrose density gradient experiments showed that both HT-BTN-α(An329) and HT-BTN-pII-α (An317) proteins were assembled into thephycobilisomes. To test whether the lack of An317 and An329biotinylation in vivo was due to the fact that the recombinantphycocyanins were quickly assembled into the phycobilisome uponbiosynthesis and therefore not available to the biotin ligase, thecyanoglobin (GIbN) protein from cyanobacterium Nostoc commune (66) wasused. This myoglobin-like monomeric hemoprotein has a specificsubcellular location around the periphery of the cytosolic face of thecell membrane (69), but is not membrane-bound. His-tagged cyanoglobinsHT-GlbN and HT-BTN-GlbN (encoded by plasmids pBS121 and pBS355,respectively; Table 7) expressed in both E. coli and Anabaena sp. werefound to have absorbance spectra nearly identical to that described forthe native GlbN (70). However, while the Ec355 protein was found to bebiotinylated, the An355 protein was not. The negative results forbiotinylation in Anabaena sp. of all three different proteins tested,An317, An329, and An355, suggest that the BTN tag is not recognized bythe Anabaena biotin ligase.

[0133] Analysis of the IMAC-purified An329 proteins by SDS-PAGE incatedthe composition HT-BTN-α:β. The An329 SEC-HPLC elution profile andspectroscopic characteristics were virtually identical to thenon-BTN-tagged counterpart An168 (HTα:β; Table 8). The yield, however,was surprisingly low; An329 recovered by IMAC was <3% of total cellularphycobiliprotein, over 10-fold less than obtained for An168. This wasalso the case for An317 compared to its non-BTN-tagged counterpart An314[HT-pII-α:β; (Example B)]. The yield of An355 was also dramaticallylower than that of An121. These results suggest that the BTN tagdestabilizes the tag-bearing proteins in Anabaena cells. Since An317 andAn329 are assembled into the phycobilisomes, the latter may also bedestabilized. Higher turnover of phycobilisomes and of An329 proteins inthe cell may explain the observed phenotype of lower cellularphycobiliproteins in the strains Anabaena sp. PCC7120(pBS317) andAnabaena sp. PCC7120(pBS329).

[0134] Expression of truncated Anabaena BCCP114 protein—Since the BTNtag was not biotinylated in Anabaena sp., naturally occurringbiotinylated proteins were investigated. A truncated gene encoding theC-terminal 114 residues of the Anabaena BCCP protein, covering thebiotinylation domain (corresponding to the E. coli BCCP84 that issufficient for enzyme-catalyzed biotinylation) and a large portion ofthe flexible linker, was amplified from the genome and cloned intopBS350, giving plasmid pBS344 (Table 7). Plasmid pBS344 encodes the6xHis-tagged Anabaena BCCP114 with a long linker bearing the TEV andthrombin protease sites.

[0135]E. coli cultures expressing the 6xHis-tagged Anabaena BCCP114protein (Ec344) grew normally, and produced very soluble Ec344 proteinat >10 mg per liter of culture grown and induced at 37° C. The proteinwas shown biotinylated by Western analysis. Electrospray massspectroscopy analysis suggested that about 20% of the Ec344 proteinproduced under these particular conditions was biotinylated. Growth andinduction at 30 ° C. for longer period of time was attempted to increasethe percentage of biotinylated holoprotein fraction of purified Ec344.However under these conditions, the yield of affinity purified proteinwas greatly decreased, possibly as a result of lowered production andincreased degradation of the Ec344 protein.

[0136] Cultures of Anabaena sp. PCC7120(pBS344) expressing the An344protein were fairly healthy, albeit having a slightly reduced level oftotal cellular phycobiliproteins. The yield of An344 protein, however,was quite low (usually <1 mg per liter of dense culture), possibly as aresult of proteolytic clipping of the relatively long linker between the6xHis tag and the BCCP114 domain. The An344 protein was shown to bebiotinylated in Western analysis. Electrospray mass spectral analysisindicated that about 40% of the molecules were holo- (i.e.,biotinylated) proteins, substantially higher than the fraction of thoseproduced in E. coli. These results encouraged the study of C-terminalphycocyanin α and β fusions with BCCP 114.

[0137] Expression of phycocyanin β subunit-BCCP114 fusionproteins—Plasmid pBS353 encodes the fusion protein HTβ-BCCP114 (Table7). Overexpression of Ec353 in E. coli gave high yield of the protein insoluble form. Since expression of Ec262 (HTβ) under similar conditionsleads to substantial formation of inclusion bodies (Example A), theBCCP114 domain appears to enhance the solubility and folding of thefusion protein. The HTβ-BCCP 114 preparation from E. coli was foundbiotinylated in Western analysis.

[0138] Cultures of Anabaena sp. PCC7120(pBS353) generally had anunhealthy, yellowish green appearance, and the growth rate was at least20% slower than that of the wild type even under high-light conditions.The yellowish green color of the cultures results from ˜50% reduction ofcellular phycocyanin as indicated by whole-cell absorption spectra. UponIPTG induction of overproduction of the HTβ-BCCP114 protein, cultures inlate-log growth frequently stopped growing, and further incubation ofthe culture often led to general cell lysis. This unexpected result,which was also observed with the HTα-BCCP114 construct (An351; seebelow), led to the abandonment of IPTG induction of Anabaena sp.PCC7120(pBS353) cultures. Expression of the HTβ-BCCP114 protein,therefore, was left to the leaky trc promoter (Example A), and generallyresulted in a fairly low yield of An353 (<5% of total cellularphycobiliproteins). Nonetheless. very high cell densities could beobtained with these uninduced cultures.

[0139] The HTβ-BCCP114 protein was incorporated into phycobilisomes,with no obvious destabilizing effects. On SDS-PAGE analysis, however,the affinity-purified An353 protein had a substantially larger amount ofthe HTβ-BCCP114 subunit than the partner phycocyanin holo-α subunit,indicating a lowered stability of α:HTβ-BCCP114 as compared with that ofα:HTβ. Also, a small fraction of the HTβ-BCCP114 preparation had lostthe BCCP114 moiety by proteolysis.

[0140] On SEC-HPLC the An353 preparation separated into two majorcomponents. The larger component, with a retention time of about 12.4min, had an apparent mass of 183.7 kDal, somewhat larger than 162.2 kDalcalculated for the trimer, (α:HTβ-BCCP114)₃. On SDS-PAGE this largerfraction had only phycocyanin α and HTβ-BCCP114 subunits, in nearly 1:1ratio. Upon exposure to Zn²⁺, fluorescence from the HTβ-BCCP114 bandunder UV illumination was about twice as bright as that from thephycocyanin holo-α subunit, suggesting full bilin content (two PCBs perβ) of the recombinant phycocyanin β subunit. The smaller component,eluted at ˜14.4 min, contained mostly HTβ-BCCP114, with an apparentmolecular weight corresponding to that of the homodimer, (HTβ-BCCP114)₂.While this result is in accord with the observation that phycocyanin HTβsubunits can form stable homodimers (Example A), it also suggests thatthe HTβ-BCCP114 subunits have a lowered affinity for the phycocyanin αsubunit, and may lose some of the α subunits during affinitypurification.

[0141] Polypeptides in the HTβ-BCCP114 preparation lacking the BCCP114portion represent a higher proportion of the fraction containing(HTβ-BCCP114)₂ than of the (α:HTβ-BCCP114)₃ fraction. Such proteolyticremoval of the BCCP114 domain of Ec353 was not observed in E. coli. Thedegradation observed in the preparations from Anabaena cells maytherefore be the result of the much longer time the fusion protein An353remains inside Anabaena cells, and may also reflect higher activity ofAnabaena sp. proteases towards the long, thrombin site-bearing linker.

[0142] Spectroscopic properties of the trimeric fraction,(α:HTβ-BCCP114)₃, were similar to those of (α:HTβ)₃ (Table 8). Theslightly blue-shifted λ_(mα) of (α:HTβ-BCCP114)₃ and the lowerA_(max):A_(360nm) ratio may reflect incomplete bilin addition to the βsubunit, with a consequent perturbed conformation and lowered trimerstability in a portion of the preparation.

[0143] Western analysis showed that HTβ-BCCP114 produced in Anabaena sp.was biotinylated. Binding experiments with monomeric avidin-coated beadswere performed to further assess the utility of An353 proteins aslabels. When avidin molecules were in excess. ˜30% of the An353preparation could be immobilized on the beads and then specificallyeluted. indicating a level of HTβ-BCCP114 biotinylation in Anabaena sp.of about 30%. When an excess of An353 was used, saturation by therecombinant phycocyanin of nearly all of the biotin-binding sites on theavidin-coated beads was achieved. With appropriate excitation, thestained beads emitted brilliant red fluorescence.

[0144] Expression of HTβ-BCCP114fusion protein bearing the GCN4-pIItrimerizattion domain —Only about a half of the HTβ-BCCP114 expressed inAnabaena retained the phycocyanin α subunit during purification. We hadnoted in earlier studies (Example B) that phycocyanin fusion proteinswith GCN4-pII trimerization domains remained trimeric even at very lowprotein concentrations and contained both subunits in stoichiometricamounts. This is most likely attributable to the greatly increased localconcentration of subunits.

[0145] HT-pII-β-BCCP114, encoded by plasmid pBS359 (Table 7), wasexpressed at high level in E. coli. About 25% of Ec359 could be isolatedin soluble form, much more than seen with pII-phycocyanin subunit fusionconstructs (Example B), again showing the increase in solubilityattributable to the presence of the BCCP114 domain (see above).

[0146] Cultures of Anabaena sp. PCC7120(pBS359) were yellowish, similarto those of Anabaena sp. PCC7120(pBS353), with an even slower growthrate. However, such cultures grew to very high cell densities after IPTGinduction.

[0147] The HT-pII-β-BCCP114 protein was assembled into phycobilisomeswith no obvious destabilizing effect. In contrast to An353,affinity-purified An359 protein preparation had the HT-pII-β-BCCP114subunit and the phycocyanin holo-α subunit in a 1:1 ratio. The yield olAn359 was surprisingly low, generally <2% of total cellularphycobiliproteins. Almost no HT-pII-β lacking the BCCP114 moiety wasobserved, suggesting that in the stable trimers. (α:HT-pII-β-BCCP114)₃,the long linker between β and BCCP114 is shielded from proteases.

[0148] On SEC-HPLC, the An359 protein preparation migrates as acomponent with an apparent mass of 475.2 kDal, significantly higher thanthe 350.3 kdal calculated for a hexamer, [(α:HT-pII-β-BCCP114)₃]₂. Aphycocyanin hexamer is normally formed by face-to-face stacking of twotrimers (42). A model of the (α:HT-pII-β-BCCP114)₃ trimer reveals that ahexamer could form by two trimers stacking on the BCCP114 face, with theBCCP114 domains on the outside of the trimer rings. Such a hexamer wouldhave a larger radius of gyration and exhibit a higher apparent mass onSEC. It should be noted that at very low protein concentration thehexamer is expected to dissociate into trimers. Spectroscopic propertiesof (α:HTβ-BCCP114)₃ (Table 8), were very similar to those ofGCN4-pII-bearing constructs such as (HT-pII-α:β)₃.

[0149] Western analysis showed that HT-pII-β-BCCP114 obtained fromAnabaena sp. was biotinylated. The utility of this construct as afluorescent label was explored in binding experiments with avidin-coatedbeads. With avidin in excess, about 75% of (α:HT-pII-β-BCCP114)₃ wasimmobilized on the beads and then specifically eluted off. The extent ofbiotinylation of the BCCP114 domain in HT-pII-β-BCCP114 proteins ispresumably ˜30%. similar to that seen in HTβ-BCCP114 (An353). The higherbinding percentage is anticipated. since only one of the three BCCP114domains needs to be biotinylated for the entire trimer to bind to thebeads. When an excess amount of (α:HT-pII-β-BCCP114)₃ was used, theamount of α:HT-pII-β-BCCP114 monomer equivalents immobilized was nearly2.5-fold that of monomeric avidins. Under phycocyanin excitation,(α:HT-pII-β-BCCP114)₃-stained avidin- and streptavidin-coated beads werehighly fluorescent.

[0150] Expression of phycocyanin-BCCP114 fusion protein with covalentlybridged α and β subunits—Constructs with covalently bridged α and βsubunits provide a way of ensuring 1:1 α:β stoichiometry, asdemonstrated earlier with the HTα-L11-β construct (Example B).HTα-L11-β-BCCP114 encoded by plasmid pBS361 (Table 7) was expressed wellin E. coli and acceptably in Anabaena sp.

[0151] HTα-L11-03-BCCP114 protein was assembled into the phycobilisome.As with HTα-L11-β (Example B), such phycobilisomes were less stable. Theaffinity-purified An361 fraction represented ˜10% of total cellularphycobiliprotein and consisted >90% of HTα-L11-β-BCCP114. A small amountof native phycocyanin α and β subunits copurified withHTα-L11-β-BCCP114, indicating that the α and 62 subunits in the fusionconstruct retained their ability to interact with native subunits. Asmall fraction of the HTα-L11-β-BCCP114 proteins had lost the BCCP114portion by proteolysis.

[0152] SEC-HPLC fractionation showed that the An361 preparationconsisted of trimers and monomers in an ˜1:1 ratio. Spectroscopicproperties of the trimers, (HTα-L11-β-BCCP114)₃, were found very similarto those of (HTα-L11-β)₃ (Table 8), indicating that carbα yl terminalattachment to BCCP114 did not perturb the phycocyanin subunit domains.In a related construct. involving a carbα yl terminal fusion of the αsubunit, (HTα-L11-β)₃, bilin addition to the α subunit domain was shownto be very incomplete (Example B). Since (HTα-L11-β)₃ and(HTα-L11-β-BCCP114)₃ have virtually identical spectroscopic properties(Table 8), bilin addition to the α subunit domain of(HTα-L11-β-BCCP114)₃ is presumably also incomplete.

[0153] The HTα-L11-β-BCCP114 protein preparation from Anabaena sp. wasshown to be biotinylated in Western analysis. In binding experimentswith streptavidin- and avidin-coated beads, the An361 preparation gaveresults similar to those obtained with HTβ-BCCP114 (see above),indicating ˜30% biotinylation of the BCCP domain of HTα-L11-β-BCCP114.

[0154] Expression ofphycocyanin-BCCP114 fusion protein with covalentlybridged α and β subunits and the GCN4-pII trimerization domain—PlasmidpBS365 was constructed to encode the HT-pII-α-L11-β-BCCP114 (Table 7).Unlike Ec362 (HT-pII-α-L11-β) which is found almost entirely ininclusion bodies (Example B), Ec365 expressed in E. coli remainedsoluble, again indicating the solubility-promoting effect of the BCCP114domain.

[0155] HT-pII-α-L11-β-BCCP114, expressed in Anabaena sp.PCC7120(pBS365), was found to be assembled into the phycobilisome. Theyield of affinity-purified An365 was >5% of total cellularphycobiliprotein. The An365 preparation consisted of >90%HT-pII-α-L11-β-BCCP114 with a small amount of copurifying nativephycocyanin α and β subunits. Virtually no loss of the BCCP114 moietywas observed.

[0156] On SEC-HPLC, the An365 preparation was found to consist mostly ofhexamers, with a small fraction of trimers. (HT-pII-α-L11-β-BCCP114)₃had spectroscopic properties similar to those of (HT-pII-α-L11-β)₃(Table 8), again consistent with incomplete bilin addition to thephycocyanin α domain (Example B).

[0157] The An365 from Anabaena sp. was shown to be biotinylated inWestern analysis and gave results in binding experiments with monomericavidin-coated beads quantitatively similar to those obtained with An359,(α:HT-pII-β-BCCP114)₃. Streptavidin-coated beads stained with excess ofAn365 appeared blue and emitted brilliant red fluorescence underappropriate excitation.

[0158] Expression of phycocyanin α subunit-BCCP114 fusionproteins—Recombinant phycocyanin α subunits, with the amino-terminusfused to polypeptides of varying length, display unmodified bilincontent and spectroscopic properties (Example A, Example B). As notedabove, fusions at the carbα yl terminus of the α subunit interfered withbilin addition. To examine whether this behavior is specific toparticular C-terminal fusions or is general, four phycocyanin α-BCCP114fusion constructs analogous to the β-BCCP fusions described above wereprepared (Table 7).

[0159] Expression of the four constructs, Ec351, Ec357, Ec360, andEc364, in E. coli gave results very similar to those observed with thefour corresponding phycocyanin β-BCCP114 constructs (see above).Expression of the four α-BCCP114 constructs in Anabaena sp., however.gave very different results.

[0160] An351 (HTα-BCCP114) represented <0.5% of total cellularphycobiliprotein. SDS-PAGE showed that the HTα-BCCP114 polypeptide had avery low bilin content, indicating that the BCCP114 extension on the αsubunit greatly interferes with bilin addition. The low yield is likelyattributable to the ease of proteolysis of the apo-HTα-BCCP114polypeptide. The An351 preparation contained little phycocyanin βsubunit, suggesting reduced affinity to the apo-HTα-BCCP114. Theproteolytic cleavage of the long peptide linker between α and BCCP114was also high in the apo-HTα-BCCP114 polypeptide. An351 preparationsoften contained a substantial amount of degradation product ofHTα-BCCP114 in which the BCCP114 moiety had been clipped off. Theextremely low yield of An351 also suggests that the protein is rapidlyturned over in the cell.

[0161] Results from expression of An357 (Table 7) were nearly identicalto those from An351, except that the native phycocyanin β subunit wascopurified with the (mostly apo-) HT-pII-α-BCCP114 subunit in nearly 1:1ratio. Results from expression of An360 (HTβ-L11-α-BCCP114) and An364(HT-pII-β-L11-α-BCCP114) were very similar. The yield was ˜5% of totalcellular phycobiliprotein, substantially higher than that obtained fromAn351 and An357. A large percentage of the purified fusion protein,however, had lost the BCCP114 moiety. These results showed thatinterference with bilin addition to the phycocyanin α-subunit domain wasa feature common to all of these different BCCP114 fusion constructs.

[0162] Expression of phycocyanin α subunit-Strep tag fusion proteins—Toaddress the question of the possible dependence of bilin addition to thephycocyanin α subunit on the size of the carbα yl-terminal fusionpartner, such fusions were prepared with the 10-residue Strep tag (Table7).

[0163] The yield of the His-tagged protein preparation, An386, fromAnabaena sp. PCC7120(pBS386) expressing HTα-Strep was <1% of totalcellular phycobiliprotein (in great contrast to >30% usually obtainedfrom cells expressing HTXα). On SEC-HPLC the An386 preparation runsalmost entirely as monomer, with a very small amount of trimers. Themonomer peak contained HTα-Strep and native phycocyanin β in nearly 1:1ratio. While the β subunit was the normal holoprotein, the HTα-Strepsubunits were almost entirely apo-subunits. The apo-HTα-Strep appearedto have lowered affinity for the phycocyanin β subunit, as indicated bythe observation that fractions collected from the trailing part of themonomer peak on SEC-HPLC were enriched in the apo-HTα-Strep subunitrelative to the holo-β subunit. The spectroscopic properties of theHTα-Strep:β monomer were very similar to those of the apo-HTα:holo-βmonomer (Table 8; Example A), including the characteristic broad peak inthe fluorescence excitation spectra. Thus, even a short extension on thecarbα yl terminus of phycocyanin α subunit affects the posttranslationalbilin addition.

[0164] Expression of phycocyanin fusion protein with covalently bridgedα and β subunits and the Strep tag—In a final attempt to address theproblem of incomplete bilin addition in carbα yl terminal fusions of thephycocyanin α-subunit, we examined behavior of HTβ-L11-α-Strep fusions(encoded by plasmid pBS394; Table 7). The yield of affinity-purifiedAn394 was ˜10% of total cellular phycobiliprotein. Unlike the An315preparation (from cells expressing HTβ-L11-α) that consists mostly oftrimers, the An394 proteins is mostly monomeric, with small amounts oftrimeric and hexameric components. Proteins in the higher assemblystates had a higher bilin content, with those in the hexamer fractionapproaching three PCBs per HTβ-L11-α-Strep molecule, i.e., holoproteins.Proteins in the monomer fraction on average contained two PCBs perHTβ-L11-α-Strep molecule, most likely lacking the α subunit bilin. Intotal, <10% of the polypeptides in the An394 preparation carried bilinon the α subunit domain, a small but significant improvement over theHTα-Strep:β construct. Most An394 preparation could be immobilized onstreptavidin-coated beads, showing that the Strep tag in theHTβ-L11-α-Strep molecules has retained its affinity for streptavidin andis available for binding. TABLE 1 Properties of Anabaena sp. PCC7120 andderivative strains and of expression vectors carried by these strainsrelevant to the production of His-tagged C-phycocyanin α and β subunitsStrain genomic Polypeptide construct Strain and vector^(a)characteristics encoded on vector PCC7120(pBS168) Wild-type HTαPCC7120(pBS262) Wild-type HTβ B646(pBS168) cpcBAC^(b) has a transposoninserted HTα between the promoter and the cpcB open reading frameB646(pBS262) cpcBAC HTβ B64328(pBS168) cpcE^(c) inactivated bytransposon HTα insertion B64328(pBS262) cpcE HTβ B64407(pBS168) cpcF^(c)inactivated by transposon HTα insertion B64407(pBS262) cpcF HTβPCC7120(pBS167) Wild-type HTα^(A12T) PCC7120(pBS162) Wild-typeHTβ^(S46G,N76D)

[0165] TABLE 2 Molecular weights of His-tagged phycocyanins in differentaggregation states, as determined by size exclusion chromatography andby analytical ultracentrifugation. SEC-HPLC AUC Major AggregationExpected Protein^(a) (kDa) (kDa) Components state MW (kDa) Native PC211.7 α:β Hexamer 225.7 124.7 Trimer 112.8 44.5 Monomer 37.6 An168 301.6HTα:β Hexamer 243.0 145.3 125.9 Trimer 121.5 49.2 34.8 Monomer 40.5An262 136.9 121.9 α:HTβ Trimer 121.5 62.4 43.4 Monomer 40.5 An168-BAC289.3 HTα:β Trimer 243.0 137.2 Monomer 121.5 17.3 HTα Subunit monomer20.9 An262-BAC 50.3 HTβ Subunit homodimer 45.0 An168-E/F 106.8 Apo-HTα:βTrimer 119.7 29.8 Monomer 39.9 An262-E/F 54.1 HTβ Subunit homodimer 45.033.8 Apo-α:HTβ Monomer 39.9

[0166] TABLE 3 Spectroscopic properties of His-tagged phycocyanins^(a).Abs^(max) Ex^(max) Em^(max) εm (M⁻¹cm⁻¹) Sample (nm) (nm) (nm) (x1,000)Φ_(f) Native PC (α:β)₃ 615-618 615 644 929 ± 2.1 0.27 ± 0.06 (HTα:β)₃615-621 618 642 921 ± 36 0.22 ± 0.05 (α:HTβ)₃ 615-619 616 642 888 ± 330.23 ± 0.04 HTα 618-622 615 637 109 ± 2.5 0.23 ± 0.02 (HTβ)₂ 605 606 639365 ± 13 0.22 ± 0.03 apo-HTα:β 607 606 642 182 ± 1.4 0.24 ± 0.00 Oneprotein- 660^(b) — — 35.40 0 bound PCB 280 — — 14.85 ± 0.1

[0167]^(a) Absorbance maxima (Abs^(max)) are for protein concentrationsbetween 0.05 and 3 μM. Lower concentrations yield blue-shifted spectrain all cases where a range of wavelengths is given. Excitation andemission maxima (Ex^(mα) and Em^(max)), and quantum yields offluorescence (Φ_(f)) are for samples at ≧0.05 μM. Molar extinctioncoefficients (ε_(m)) were determined for the following complexespurified by SEC-HPLC: native PC, trimer fraction; HTα:β, trimer fractionof An168; α:HTβ, trimer fraction of An262; HTα, subunit monomer fractionof An168-BAC; HTβ, subunit homodimer fraction of An262-BAC; apo-HTα:β;monomer fraction of An168-F.

[0168]^(b) Absorbance of protein-bound phycocyanobilin was measured withSEC-HPLC purified (HTα:β)₃ and (α:HTβ)₃ holoproteins, denatured in 8 Murea pH 2.0. The molar extinction coefficient at 660 nm is identical tothat measured in 7.2 M urea pH 2.0, 9 mM DTT (35), while that at 280 nmwas calculated by subtracting contributions from Tyr [ε=1,370 M⁻¹ cm⁻¹;(36)] and Trp [ε=5,500 M⁻¹ cm⁻¹; (37)] residues. TABLE 4 Expressionplasmids with specific functional domains in the encoded protein CoiledAffinity Biospec. coil Protease Core Plasmid^(a) tag tag^(b) domain siteprotein pBS311 6xHis GCN4pII TEV LacZ^(α) pBS314 6xHis GCN4pII TEV CpcApBS319 6xHis TEV CpcA-L9-CpcB pBS320 6xHis TEV CpcA-L11-CpcB pBS3626xHis GCN4pII TEV CpcA-L11-CpcB pBS315 6xHis TEV CpcB-L11-CpcA pBS3586xHis GCN4pII TEV CpcB-L11-CpcA pBS283 Strep2 TEV LacZ^(α) pBS342 6xHisStrep2 TEV LacZ^(α) pBS327 6xHis Strep2 TEV CpcA pBS303 Strep2 GCN4pLITEV LacZ^(α) pBS309 6xHis Strep2 GCN4pLI TEV LacZ^(α) pBS323 6xHisStrep2 GCN4pLI TEV CpcA pBS312 6xHis GCN4pLI TEV LacZ^(α) pBS321 6xHisGCN4pLI TEV CpcA

[0169] TABLE 5 Determination of apparent molecular weight of componentsin recombinant phycocyanin preparations by SEC-HPLC^(a) Calc. massPercent Assembly Theoretical Protein (kDa) area state^(b) mass (kDa)^(c)An168 49.2  5 Monomer 40.5 (HTα:β) 145.3 95 Trimer 121.5 An327 44.2  4Monomer 41.4 (HT-Strep2-α:β) 137.9 94 Trimer 124.3 275.4  2 Hexamer248.7 An314 110.2 79 Trimer 132.7 (HT-pII-α:β) 406.0⁴ 21 (Hexamer)₂530.7 An321 232.8 13 Tetramer 176.9 (HT-pLI-α:β) 499.5^(d) 81(Tetramer)₃ 530.7 An323 264.0  6 Tetramer 182.7 (HT-Strep2-pLI-α:β)429.3d 94 (Tetramer)₃ 548.2 An319 35.8 79 Monomer 41.0 (HTα-L9-β) 99.319 Trimer 122.9 232.6  2 Hexamer 245.8 An320 34.1 49 Monomer 41.1(HTα-L11-β) 89.7 46 Trimer 123.4 213.6  5 Hexamer 246.7 An362 128.9 84Trimer 134.5 (HT-pII-α-L11-β) 304.7^(d) 15 Hexamer 269.1 An315 107.9 76Trimer 123.4 (HTβ-L11-α) 235.7 23 Hexamer 246.7 An358 115.6 64 Trimer134.5 (HT-pII-β-L11-α) 587.2^(d) 35 (Hexamer)₂ 538.0

[0170] TABLE 6 Spectroscopic properties of phycocyanin constructs^(a)Initial λ_(max) ^(c) A_(max)/ Ex_(max) Em_(max) ε_(M) Sample^(b)assembly state (nm) A_(360 nm) (nm) (nm) (M⁻¹cm⁻¹) Φ_(f) Denatured α:β280 — — —   44,550^(d) PC 660 1.0 — —   106,200 0 Native PC (α:β)₃615-619 6.4 615 644   929,000 0.27 An168 (HTα:β)₃ 615-621 6.4 618 642  921,000 0.22 An327 (HT-Strep2- 615-619 6.4 618 642   922,000 0.23α:β)₃ An262 (α:HTβ)₃ 615-619 6.3 616 642   888,000 0.23 An314(HT-pII-α:β)₃ 621-622 7.5 622 642   927,000 0.39 An321 (HT-pLI- 621-6237.5 621 643 1,231,000 0.28 α:β)₄ An323 (HT-Strep2- 621-623 7.2 621 6441,240,000 0.27 pLI-α:β)₄ An315 (HTβ-L11-α)₃ 615-622 6.2 622 643  900,000 0.21 An358 (HT-pII-β- 619-621 6.7 620 643   915,000 0.29L11-α)₃ An320 (HTα-L11-β)₃ 605-608 5.2 613 643   677,000^(e) 0.22 An362(HT-pII-α- 607-610 5.8 615 642   707,000^(f) 0.24 L11-β)₃

[0171] TABLE 7 List of fusion constructs indicating order of domainsFusion construct^(a) Plasmid^(b) 6xHis-BTN-TEV-LacZ^(α′) pBS3396xHis-BTN-TEV-CpcA pBS329 6xHis-BTN-pII-TEV-CpcA pBS3176xHis-BTN-TEV-GlbN pBS355 6xHis-TEV-GlbN pBS121 6xHis-TEV-TBN-BCCP114pBS344 6xHis-TEV-CpcB-TBN-BCCP114 pBS3536xHis-GCN4pII-TEV-CpcB-TBN-BCCP114 pBS3596xHis-TEV-CpcA-L11-CpcB-TBN-BCCP114 pBS3616xHis-GCN4pII-TEV-CpcA-L11-CpcB-TBN-BCCP114 pBS3656xHis-TEV-CpcA-TBN-BCCP114 pBS351 6xHis-GCN4pII-TEV-CpcA-TBN-BCCP114pBS357 6xHis-TEV-CpcB-L11-CpcA-TBN-BCCP114 pBS3606xHis-GCN4pII-TEV-CpcB-L11-CpcA-TBN-BCCP114 pBS364 6xHis-TEV-StreppBS370 6xHis-TEV-CpcA-Strep pBS386 6xHis-TEV-CpcB-L11-CpcA-Strep pBS394

[0172] TABLE 8 Spectroscopic properties of phycocyanin constructs^(a)Initial assembly λ_(max) A_(max) Ex_(max) Em_(max) Sample state^(b)(nm)^(c) /A_(360 nm) (nm) (nm) ε_(M)(M⁻¹cm⁻¹) Φ_(f) Denature α:β 280 — ——  44,550^(d) 0 d 660 1.0 — — 106,200 0 PC Native (α:β)₃ 615-619 6.4 615644 929,000 0.27 PC An168 (HTα:β)₃ 615-621 6.4 618 642 921,000 0.22An329 (HT-BTN-α:β)₃ 615-618 6.4 617 642 920,000 0.22 An386 HTα-Strep:β605-609 5.6 607 639 186,000 0.19 An262 (α:HTβ)₃ 615-619 6.3 616 642888,000 0.23 An353 (α:HTβ- 611-616 5.7 612 642 896,000 0.21 BCCP¹¹⁴)₃An314 (HT-pII-α:β)₃ 621-622 7.5 622 642 927,000 0.39 An317 (HT-BTN-pII-621-622 7.3 622 644 939,000 0.31 α:β)₃ An359 (α:HT-pII-β- 619-622 6.6621 643 915,000 0.29 BCCP¹¹⁴)₃ An320 (HTα-L11-β)₃ 605-608 5.2 613 643677,000^(c) 0.22 An361 (HTα-L11-β- 605-606 5.3 604 637 664,000^(e) 0.23BCCP¹¹⁴)₃ An362 (HT-pII-α-L11- 607-610 5.8 615 642 707,000^(f) 0.24 β)₃An365 (HT-pII-α-L11- 605-607 5.5 606 641 692,000^(f) 0.27 β-BCCP¹¹⁴)₃ #measurements at protein concentrations ≦0.05 μM. ε_(M) values are givenfor measurements on the components specified in column 2 at proteinconcentrations >1 μM.

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Further Examples

[0244] 1. Phycobiliproteins carrying the GCN4-pII or the GCN4-pLIcoiled-coil oligomerization domain.

[0245] These fusion proteins were found almost entirely in inclusionbodies in E. coli, but found incorporated in the phycobilisome inAnabaena sp. These constructs, as well as other examples discussedbelow, all show that proteins bearing the GCN4 oligomerization domainsare found almost entirely in inclusion bodies when expressed in E. coli,but are incorporated in phycobilisomes when expressed in Anabaena sp.Upon phycobilisome dissociation, good yields of soluble, fluorescentlytagged proteins are obtained.

[0246] 2. Streptavidin-phycocyanin α subunit fusion proteins.

[0247] The core streptavidin (stvC) gene used here encodes a StvCcorresponding to residues 16 to 133 of the mature streptavidin.Recombinant StvC with a 24-residue N-terminal extension bearing a 6xHistag and the TEV site. HT-StvC (encoded by plasmid pBS282), was expressedrelatively poorly in E. coli, with about 80% of the proteins found ininclusion bodies. The StvC-CpcA fusion protein has an 11 -residue bridgelinking the two moieties. HT-StvC-CpcA (encoded by plasmid pBS292) wasproduced at relatively high level in E. coli, but was found only ininclusion bodies.

[0248] The fusion protein An292 was well expressed in Anabaena sp.Ni²⁺-NTA-purified soluble An292 protein accounted for >15% of totalcellular phycobiliproteins. The An292 protein was of HT-StvC-α:β incomposition, ran on SEC-HPLC as tetramer (HT-StvC-α:β)4 and trimer oftetramers [(HT-StvC-α:β)4]3, similar to the finding with An321 withphycocyanin α subunit bearing the tetramerization domain GCN4-pLI:(HT-pLI-α:β)4 and [(HT-pLI-α:β)4]3. An292 also had spectroscopicproperties virtually identical to those of An321, making it an excellentfluorescent label. The An292 protein was assembled into phycobilisomesand also found to bind biotin.

[0249] Like streptavidin tetramers, the (HT-StvC-α:β)4 tetramer was muchmore stable than the (HT-pLI-60 :β)4 tetramer. On room temperaturedenaturing SDS-PAGE, (HT-StvC-α:β)4 released only the β subunits andretained the (HT-StvC-α)4 aggregate. The tetramer could be broken onlyby boiling the protein before loading on SDS-PAGE. Purifiedphycobilisomes displaying StvC had a tendency to aggregate out ofsolution, likely a direct result of inter-phycobilisome linkage mediatedby tetramerization of the monomeric StvC domains displayed on PBS.Usually within 48 hrs all such phycobilisomes precipitated out ofsolution.

[0250] 3. Protein A-phycocyanin α subunit fusion proteins.

[0251] Expression of truncated protein A (SpA) in the cytosol of E. colihas been shown to give very poor yield, with particularly severeproteolysis occuring to the hinge of domains A and B. In our construct,the truncated SpA protein [denoted SpA(DABC)] contains the hinge ofdomain E. domains D, A, and B in entirety, and domain C lacking a smallportion of its hinge. Domains D. A, B and C all retain their IgG-bindingstructure. Plasmid pBS356 encodes the recombinant SpA with a C-terminal6xHis tag extending from the residual hinge region of domain C:SpA(DABC)-6xHis. The protein, Ec356, was obtained in extremely pooryield when expressed in E. coli, regardless whether the purificationmethod was denaturing (using guanidine HCl; see e.g., Example B, supra)or not, and almost no full-length Ec356 protein was seen on SDS-PAGE.Recombinant protein A with engineered hinge regions has been describedfor better expression of the SpA in E. coli. Without such extensivereengineering, E. coli is not an appropriate organism for expression andproduction of recombinant protein A.

[0252] In experiments described here, the SpA(DABC) protein is fused atits C-terminus to the phycocyanin a subunit through a 24-residue linkerbearing a 6xHis tag and a recognition and cleavage site for the specificTEV endoprotease. The fusion protein, SpA(DABC)-6xHis-TEV-CpcA, encodedby plasmid pBS349, was obtained in very poor yield when expressed in E.coli, likely a result of extensive proteolysis. Little full-length Ec349protein was seen, with most of the Ni2+-NTA-purified protein having someor all of the IgG-binding domains missing.

[0253] When expressed in Anabaena sp., the fusion protein An349 wasfound assembled into phycobilisomes with no apparent destabilizingeffect on the light-harvesting complex. Cellular phycobiliprotein levelrelative to chlorophyll α, however, was about 20% lower, suggesting anelevated phycobiliprotein turnover. Nonetheless, growth rate of culturesunder high-light intensity was not affected. Ni2+-NTA-purified An349proteins were obtained in relatively low yield of about 5% of totalcellular phycobiliproteins.

[0254] On SEC-HPLC, the An349 proteins fractionated into twocomponents: >80% as the trimer (SpA-6xHis-α:β)3 and the rest beingSpA-6xHis-α subunit. SDS-PAGE showed that the SpA moiety of the majorityof the fusion protein had also been proteolyzed, giving full-lengthSpA(DABC)-6xHis-α (49.5 kDal; ca. 10%), SpA(ABC)-6xHis-α (41.5 kDal; ca.20%), and SpA(BC)-6xHis-α (35.5 kDal; ca. 70%). No proteolytic cleavageon domain B hinge [giving SpA(C)-6xHis-α] and domain C hinge (giving6xHis-α) was observed. The lack of proteolytic cleavage on the domain Bhinge is a clear indication of different protease activities in Anabaenasp. because that hinge region has been found to be the most susceptibleto proteolytic cleavage in E. coli. The lack of clipping in the domain Chinge is likely due to its prα imity to the phycobilisome, minimizingaccess by proteases. This hinge region is susceptible to proteolyticactivities in Anabaena sp. when displayed farther away from thephycobilisome surface (see below).

[0255] SDS-PAGE also showed that the carrier phycocyanin a domain hadthe normal bilin content. The SEC-HPLC trimer (SpA-6xHis-α:β)3 fractionhad spectroscopic properties very similar to those of (6xHis-α:β)3, withabsorbance maximum at 616 nm, an A616 nm:A360 nm ratio of 6.1,fluorescence excitation maximum at 618 nm (for 650 nm emission), andfluorescence emission maximum at 641 nm (for 560 nm excitation).

[0256] An349 proteins were tested for their ability to bind IgG inELISA. Serially diluted An349 protein was immobilized on a 96-well plateand then allowed to bind to mouse IgG-alkaline phosphatase conjugate.After thorough washing, the alkaline phosphatase activity was assayed bycatalyzed color development. In such semi-quantitative assays theSpA-6xHis-α:β fusion protein had about 30% IgG-binding activity ascompared to commercially obtained protein A (Sigma Chemical Co.) withall five IgG-binding domains.

[0257] Another SpA-phycocyanin a fusion protein was constructed toenhance the fusion protein's utility. This construct is similar to theSpA(DABC)-6xHis-TEV-CpcA construct described above, but has the33-residue GCN4-pII trimerization domain inserted between the 6xHis tagand the TEV site. This fusion protein, SpA(DABC)-6xHis-pII-TEV-CpcA,encoded by plasmid pBS354, was found only in inclusion bodies whenexpressed in E. coli. Unlike Ec349. about 10% of the Ec354 proteinsaffinity-purified from inclusion bodies was full-length[SpA(DABC)-6xHis-pIl-TEV-CpcA], suggesting fast sequestering of thenewly synthesized polypeptides into inclusion bodies due to bundling bythe GCN4-pII domain. The majority of purified Ec354 proteins, however,were still missing some or all of the SpA IgG-binding domains, againillustrating protein A's susceptibility to E. coli proteases.

[0258] When expressed in Anabaena sp., the fusion protein An354 wasfound assembled into phycobilisomes with no apparent destabilizingeffect on the complex. Interestingly, cells expressing An354 had a morenormal phenotype than those expressing An349 (see above).Ni2+-NTA-purified An354 proteins were obtained in reasonable yield,accounting for >7% of total cellular phycobiliproteins.

[0259] On SEC-HPLC, the An354 protein ran as a single peak with anapparent molecular weight corresponding to a hexamer,[(SpA-6xHis-pII-α:β)3]2. SDS-PAGE confirmed the composition asSpA-6xHis-pII-α:β, and showed that most of the SpA moiety was subject toproteolytic clipping at different hinge regions, giving full-lengthSpA(DABC)-6xHis-pII-α (53.2 kdal; ca. 10%), SpA(ABC)-6xHis-pII-α (45.2kDal; ca. 20%), SpA(BC)-6xHis-pII-α (39.2 kDal: ca. 50%), and6xHis-pII-α (25.2 kdal; ca. 20%). The complete removal of the displayedSpA protein by cleavage in the domain C hinge is not seen with the An349protein, and is likely a result of the SpA moiety in An354 beingdisplayed farther away from the phycobilisome surface. As with An349, noproteolytic cleavage on domain B hinge [giving SpA(C)-6xHis-pII-α] wasobserved in Anabaena sp. Different fractions collected from the An354SEC-HPLC peak had identical distributions of the fusion proteins missingvarious IgG-binding domains, consistent with the view that the fusionproteins are displayed on the phvcobilisome as monomers (with respect tothe GCN4-pII domain), and only form GCN4-pII-bundled stable trimersafter cell lysis and phycobilisome dissociation.

[0260] SDS-PAGE also showed that the carrier phycocyanin a domain hadthe normal level of phycocyanobilin. The SEC-HPLC hexamer[(SpA-6xHis-pII-60 :β)3]2 fraction had spectroscopic properties verysimilar to those of (6xHis-pII-α:β)3 (see, e.g. Example C, supra), withan absorbance maximum at 621 nm, an A616 nm:A360 nm ratio of 7.5,fluorescence excitation maximum at 621 nm (for 650 nm emission), andfluorescence emission maximum at 642 nm (for 560 nm excitation). Thesefavorable spectroscopic properties of such GCN4-pII-bundled stabletrimers make the fusion protein a good fluorescent tag. ELISA alsoshowed that An354 had IgG-binding affinity comparable to commerciallyobtained protein A. The increased IgG-binding activity of An354 in ELISAcompared to An349 may be a result of more protein being immobilized onthe 96-well plate, and/or cooperative binding of GCN4-pII-bundled SpAproteins. Such oligomerization-induced binding affinity increase hasbeen observed (see e.g.. Examples B and C, supra).

[0261] 4. Cyanoglobin fusion to phycocyanin α or β subunit.

[0262] Cyanoglobin (GlbN) is a 15.7-kDal monomeric hemoprotein producedin the cyanobacterium Nostoc commune. This protein contains anon-covalently linked heme, and is well expressed as soluble protein inboth E. coli (expression at 30° C. and 37° C.) and Anabaena sp. whenbearing a 24-residue N-terminal extension containing a 6xHis tag and theTEV recognition and cleavage site (recombinant protein encoded byplasmic pBS121; Example C, supra). However, recombinant cyanoglobin withC-terminal extension (the stop codon is replaced by a Ser codon) wasfound unable to fold normally in E. coli. Cyanoglobin-phycocyaninsubunit fusion constructs, GlbN-6xHis-TEV-CpcA (encoded by plasmidpBS190) and GlbN-6xHis-TEV-CpcB (encoded by plasmid pBS274), gaverelativelv poor yield and were found mostly in inclusion bodies whenexpression was carried out at 37° C. At 30° C., the two fusion proteinshad somewhat different behavior. Although both were still fast degradedin E. coli (thus giving low yield), the GlbN-HTa fusion protein (Ec190)was produced in a substantial amount as soluble protein, with apparentlynormal amount of heme and an absorbance spectrum identical to that of6xHis-tagged GlbN (Ec121). The GlbN-HTh fusion protein (Ec274), on theother hand, was still found mostly in inclusion bodies, with the smallamount of soluble proteins purified largely as apoproteins (withoutheme). These results indicate that the C-terminal extensions have slowedthe folding of GlbN in E. coli, and the cyanoglobin domain in theGlbN-HTh fusion protein appears to be misfolded.

[0263] Anabaena cultures expressing either fusion proteins were healthy,showing no negative phenotypes at all. Both fusion proteins, An190 andAn274, were found assembled into phycobilisomes with no destabilizingeffect on the macrostructures, were purified in high yield (eachaccounting for >20% of total cellular phycobiliproteins) with cognatepartner subunits in 1:1 ratio, and found to have spectroscopicproperties very similar to native phycocyanin.

[0264] The cyanoglobin moiety in the fusion proteins, however, behavedquite differently. Using phycocyanin a subunit as the carrier domain(An190), the displayed GlbN domain was <30% holo when eluted off theNi2+-NTA column, and lost almost all of the bound heme upon dialysis toremove imidazole. Using the phycocyanin β subunit as the carrier domain(An274), most of the displayed GlbN domain was purified with bound heme,and the heme remained bound to GlbN after dialysis. No degradation ofthe cyanoglobin domain was observed in either An190 or An274 in Anabaenacells, suggesting that the cyanoglobin domain displayed on the N′ ofphycocyanin α subunit was misfolded but remained resistant toproteolysis. The misfolded GlbN domain appears to interfere with thecarrier domain's formation of phycocyanin trimers: on SEC-HPLC, about80% of An190 proteins ran as trimers, (GblN-HT α:β)3, while the rest ranas monomers, GblN-HT α:β, in contrast to An168 which runs only astrimers, (HT α:β)3. Like An262, (α:HTβ)3, An274 ran only as trimers,(α:GlbN-HTβ)3, on SEC-HPLC.

[0265] In sum, both fusion proteins were quickly degraded in E. coli,but well displayed on phycobilisomes in Anabaena sp. Cyanoglobindisplayed on the of phycocyanin β subunit seemed to fold better than onthe N-terminus of the α subunit, and was able to retain larger amount ofheme than the latter during purification. No degradation of thecyanoglobin domain was observed in either construct in Anabaena cells,suggesting that the cyanoglobin domain displayed on the N-terminus ofphycocyanin α subunit was misfolded but remained resistant toproteolysis.

[0266] 5. Sperm whale myoglobin fusion to phycocyanin α or β subunit.

[0267] The myoglobin from sperm whale (SWMβ) is similar to cyanoglobinas a monomeric protein with non-covalently bound heme. SWMβ with aC-terminal extension of only six His residues (SWMβ-6xHis; encoded byplasmid pBS114) can be well expressed as soluble holoprotein in both E.coli and Anabaena sp. Recombinant SWMβ proteins with larger C-terminalextensions, however, appear to be unable to fold.SWMβ-6xHis-TEV-LacZ^(a) (encoded by plasmid pBS261), SWMβ-6xHis-TEV-CpcA(encoded by plasmid pBS271), and SWMb-6xHis-TEV-CpcB (encoded by plasmidpBS318) fusion proteins all gave poor yields when expressed in E. coliand were found nearly entirely in inclusion bodies.

[0268] Anabaena cultures expressing either An271 (SWMβ-HTα) or An361(SWMβ-HTβ) fusion proteins were healthy, and the fusion proteins werefound assembled into phycobilisomes, and were purified with cognatepartner phycocyanin subunits. Although myoglobin displayed on theN-terminus of phycocyanin β subunit (SWMβ-HTβ) gave slightly betteryield of the full length protein (with little amount of bound heme),both fusion proteins were purified mostly without the myoglobin moiety.This indicates that the displayed domain, if unable to fold, isdegraded.

[0269] 6. Maltose-binding protein (MalE, MBP) fusion to phycocyanin α orβ subunit.

[0270] The “mature” recombinant MalE protein (lacking themembrane-crossing signal peptide) with a C-terminal 6xHis tag is wellexpressed as a soluble protein in the cytosol of both Anabaena sp. andE. coli. In the MalE-phycocyanin fusion constructs, the last threeresidues (Arg-Ile-Thr) of the mature MalE are replaced by the spacerAsn-Ser-Ser, and this modified MalE is fused to the N-teiminus of aphycocyanin subunit via a 24-residue linker that bears a 6xHis tag and arecognition and cleavage site for the TEV protease. The MalE-6xHis-TEV-α(Ec396) and MalE-6xHis-TEV-β (Ec398) fusion proteins are both expressedas soluble proteins in E. coli in relatively high yield.

[0271] The strain Anabaena sp. PCC7120(pBS396) expressing fusion proteinMalE-6xHis-TEV-α had a phenotype very similar to that of strain Anabaenasp. PCC7120(pBS168) expressing HTα. Sucrose density gradientsedimentation fractionation showed that the MalE-6xHis-TEV-α fusionprotein was assembled into the phycobilisome without obvious effects onthe macromolecular complex. Ni²⁺-NTA affinity-purified protein, An396,was in relatively good yield, accounting for >10% of total cellularphycobiliproteins. Over 95% of the An396 proteins can bind to, and bespecifically eluted from amylose columns, diagnostic of functional MalE.

[0272] SDS-PAGE showed that the An396 protein has the compositionMalE-6xHis-TEV-α:β, and Zn2+-induced fluorescence indicates fullchromophorylation of phycocyanin carrier domain. SEC-HPLC showed thatAn396 protein was monomeric, (MalE-6xHis-TEV-α:β) a behavior differingfrom that of HTα:β, whose elution volume is that of a trimer. TheMalE-6xHis-TEV-α:β protein in the SEC-HPLC monomer peak fraction,however, has spectroscopic properties very similar to the (HTα:β)3trimer, with absorbance maximum at 617 nm, fluorescence excitationmaximum at 618 nm (for 650 nm emission), fluorescence emission max. at637 nm (for 560 nm excitation), and a fluorescence quantum yield of0.23.

[0273] Upon digestion with the TEV protease, over 90% of purifiedMalE-6xHis-TEV-α:β protein can be clipped at the TEV recognition site toseparate the displayed domain (MalE) from the phycocyanin carrierdomain.

[0274] The fusion protein MalE-6xHis-TEV-β is expressed in Anabaena sp.at a level (>7.5% of total cellular phycobiliproteins) comparable tothat of MalE-6xHis-TEV-α (An396). Over 95% of Ni2+-NTA-purified An398protein can bind to, and then be specifically eluted from, the amyloseresin, indicative of the MalE function. However, analysis of An398 bySDS-PAGE shows that the protein preparation consists mostly ofMalE-6xHis-TEV-β with only a very small amount of the partnerphycocyanin α subunit. Zn2+-induced fluorescence suggests that theMalE-6xHis-TEV-β protein has a full complement of phycocyanobilin, i.e.,two PCBs per β subunit domain. On SEC-HPLC most of the preparation runsas (MalE-6xHis-TEV-β)2 homodimer, with a slight shoulder correspondingto the (α:MalE-6xHis-TEV-β) monomer. Like MalE-6xHis-TEV-α:β (An396),virtually all of the MalE-6xHis-TEV-β fusion proteins can be cleaved atthe TEV site to separate the displayed protein (MalE) and the carrierprotein (phycocyanin β subunit).

[0275] The MalE-6xHis-TEV-β protein is found to be assembled intophycobilisomes without obvious deleterious effect on the latterstructures. Since assembly of the fusion protein into the phycobilisomesrequires association with the phycocyanin α subunit, this resultindicates that the MalE-6xHis-TEV-β fusion protein has a reducedaffinity to the α subunit which is likely lost during the purificationprocess. In the absence of α subunits, phycocyanin β subunits have beenshown to form stable homodimers (see. e.g. Example A, supra). Suchinterference by the displayed domain on the phycocyanin subunit carrierdomain's affinity for its cognate partner subunit is likely fusionprotein specific—cyanoglobin displayed on the N-terminus of thephycocyanin β subunit does not appear to have obvious effects on thefusion protein's affinity for the phycocyanin α subunit.

[0276] The (MalE-6xHis-TEV-β)2 homodimer peak fraction in SEC-HPLC wasused for spectroscopic characterization. Similar to the (HTβ)2 homodimerprotein, the (MalE-6xHis-TEV-β)2 homodimer has absorbance andfluorescence excitation maxima at 605 nm, a fluorescence emissionmaximum at 638 nm, and a fluorescence quantum yield of 0.26.

[0277] To obtain 1:1 α:β stoichiometry, the β-L11-α subunit-fusionphycocyanin (Example B. supra) can be used, in place of the β subunit,as a carrier protein. Constructs incorporating the GCN4-pIItrimerization domain, such as (MalE-6xHis-pII-α:β)3 and(MalE-6xHis-pII-β-L11-α)3, have excellent spectroscopic properties, andare very useful, for instance, in studies of protein glycosylation.

[0278] All publications and patent applications cited in thisspecification and all references cited therein are herein incorporatedby reference as if each individual publication or patent application orreference were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A cell comprising a functional oligomericphycobiliprotein comprising a fusion protein comprising a functionaldisplayed domain and a functional phycobiliprotein domain.
 2. The cellof claim 1, wherein the phycobiliprotein domain is a naturalphycobiliprotein domain.
 3. The cell of claim 1, wherein the functionaloligomeric phycobiliprotein is an α,β heterodimer.
 4. The cell of claim1, wherein the displayed domain comprises a moiety selected from thegroup consisting of an affinity tag, an oligomerization moiety, aspecific binding moiety, and a signaling moiety.
 5. The cell of claim 1,wherein the fusion protein further comprises a specific binding moietyselected from a streptavidin biotin-binding moiety, a biotinylated orbiotinylatable moiety, and an antigen binding immunoglobulin moiety. 6.The cell of claim 1, wherein the fusion protein further comprises alinker peptide between the displayed domain and the phycobiliproteindomain.
 7. The cell of claim 1, wherein the fusion protein furthercomprises a protease cleavage site between the displayed domain and thephycobiliprotein domain.
 8. The cell of claim 1, wherein thephycobiliprotein domain comprises at least one functionally attachedbilin.
 9. The cell of claim 1, wherein the displayed domain isrefractive to expression in E. coli.
 10. The cell of claim 1, whereinthe oligomeric phycobiliprotein is assembled in a functionalphycobilisome.
 11. The cell of claim 1, wherein the oligomericphycobiliprotein provides a fluorescent tag.
 12. The cell of claim 1,wherein the displayed domain is substantially transparent to wavelengthsof visible light absorbed by phycobiliproteins.
 13. The cell of claim 1,wherein the displayed domain is substantially transparent to wavelengthsof energy emitted by the phycobiliprotein domain.
 14. The cell of claim1, wherein the cell is or is a progeny of a cell which naturallyexpresses a phycobiliprotein.
 15. The cell of claim 1, wherein the cellis a cyanobacterium.
 16. The cell of claim 1, wherein the cell is arhodophyte (red algae).
 17. The cell of claim 1, wherein the cell is acryptomonad.
 18. The cell of claim 1, wherein the cell is an Anabaenacell.
 19. The cell of claim 1, which comprises a polynucleotide encodingthe fusion protein, and produces the oligomeric phycobiliprotein.
 20. Amethod for making a functional oligomeric phycobiliprotein, comprisingthe step of incubating the cell of claim 19 to express the fusionprotein and produce the oligomeric phycobiliprotein.
 21. A method formaking a functional displayed domain, comprising the step of incubatingthe cell of claim 19 to express the fusion protein and produce theoligomeric phycobiliprotein, cleaving a peptide bond between thefunctional displayed domain and the functional phycobiliprotein domain;and separating the functional displayed domain from the functionalphycobiliprotein domain.
 22. The method of claim 21, which providesimproved functional folding of the displayed domain, as compared withexpression of the displayed domain not fused to the phycobiliproteindomain.